"Russia is failing to genuinely de-conflict airspace in Syria. Some of these incidents are not mistakes," Chief Pentagon Spokesperson Dana W. White told CNN.
"We are working to address this issue at the highest levels," she added.
The Pentagon's statement comes just days after Secretary of Defense Jim Mattis said it was unclear why the Russians were violating the agreement, which separates Russian and US-led coalition forces operating in Syria.
"I don't expect perfection, but I don't expect dangerous maneuvers, either," Mattis told reporters Friday when asked about the recent violations of the de-confliction line, suggesting that some of the violations may have been due to pilot error.
"We'll sort this out but right now, I cannot tell you if it's sloppy airmanship, or a rambunctious pilot, or people who are trying to do something that was very unwise," Mattis said.
The US military said last week that two US F-22 stealth fighters intercepted two Russian Su-25 attack jets after the Russian aircraft crossed the Euphrates River in Syria, flying east of the "de-confliction line" that is supposed to separate Russian and US-led coalition aircraft operating over Syria.
"One Su-25 flew close enough to an F-22A that it had to aggressively maneuver to avoid a midair collision," US Air Forces Central Command spokesman Lt. Col. Damien Pickart told CNN last week, saying the US planes conducted multiple maneuvers, released chaff and flares, and made "multiple calls on the emergency channel to convey to the Russian pilots that they needed to depart the area."
The Russian Ministry of Defense issued a statement last week denying its planes crossed the de-confliction line, saying the incident took place west of the Euphrates River.
President Donald Trump and Russian President Vladimir Putin pledged to continue to uphold these de-confliction measures in their joint statement issued on the sidelines of the APEC conference in Da Nang, Vietnam.
The statement said Trump and Putin "agreed to maintain open military channels of communication between military professionals to help ensure the safety of both US and Russian forces and de-confliction of partnered forces engaged in the fight against ISIS."
The agreement says that Russian and Syrian regime forces are to operate west of the Euphrates River while the US and its coalition allies are to operate to the east. If either side wishes to operate on the other side of the river, they are supposed to communicate via a hotline that allows Russian and coalition military officers to talk to each other.
Asked Friday about the Pentagon's statement, Kremlin Spokesman Dmitry Peskov referred CNN to the Russian Ministry of Defense while also accusing the US military of having no legal justification for being in Syria.
"The Russian military are present in Syria absolutely legitimately, in full compliance with the norms and principles of international law, as for the US armed forces, they are there without any international legal grounds," Peskov said.
The de-confliction arrangements gained greater importance in June after a US Navy F/A-18 shot down a Syrian regime Su-22 jet after the regime warplane dropped bombs near US-backed Syrian Democratic Forces near Tabqah, Syria.
"Since agreeing to this de-confliction arrangement, the Russians have flown into our airspace on the east side of the river 6-8 times per day, or approximately 10% of the Russian and Syrian flights," Pickart said.
But a spokesman for the US-led coalition fighting ISIS told reporters Tuesday that Russian jets were flying fewer missions alongside the Euphrates River, reducing the number of potential interactions between Russian and US/coalition aircraft.
"In the last two to three days, there has been a decrease in the presence along the Middle Euphrates River Valley," Col. Ryan Dillon said while adding "there has been a decrease in the amount of sorties that they have flown."
On Thursday, a US Air Forces Central Command spokesperson, Capt. AnnMarie Annicelli, told CNN: "We have not had an intercept since Dec. 13, 2017. There has been a marked decrease in incidents between Coalition and Russian Federation aircraft. This is due in large part to the dialogue from both sides that has allowed us to voice concerns and seek solutions that de-escalate tensions."
A spokesperson for the US-led coalition told CNN Friday that officers from the coalition met face to face with their Russian counterparts last month to discuss de-conflicting operations in Syria. Such face-to-face meetings are relatively rare, with only a handful having taken place since they began in September.
That first meeting was revealed shortly after Russian warplanes bombed US-backed anti-ISIS fighters in Syria while US military advisers were nearby.
The spokesperson also said that the commander of the coalition, US Army Lt. Gen. Paul Funk, recently exchanged emails with his Russian counterpart, Colonel-General Alexander Zhuravlev, saying that Funk welcomed Zhuravlev to his new post and that both men "discussed the importance of de-confliction."
However, a US military official told CNN that as recently as Saturday, a Russian aircraft carried out airstrikes east of the Euphrates River Valley without informing the coalition via the de-confliction hotline.
Annicelli later confirmed the incident, telling CNN that it took place on December 15 and that the coalition contacted the Russian air force via the hotline to discuss the incident.
She said the Russians did not say whether it was a Russian or Syrian aircraft that dropped the bombs but the Russian officers attributed the incident to a "mistake in target coordinates," according to Annicelli.
The breakdown of the effectiveness of the de-confliction line raises serious questions about the fight against ISIS as coalition officials have said that ISIS defenders continue to fight in remaining villages along the Euphrates River Valley while other ISIS fighters have moved west into areas ostensibly controlled by the Syrian regime.
ISIS fighters "know if they were to come east of the river that they have to contend with our partners, the Syrian Democratic Forces. So they would opt for the easier solution, which is, moving west," Dillon said.
Asked if US-led coalition forces could strike ISIS fighters further west of the Euphrates without running afoul of Russian airplanes, Dillon demurred.
"As far as going further into the western part, or the interior parts of the -- of Syria, I'm not prepared and ready to address that," Dillon said.
Amid these multiple alleged Russian crossings of the de-confliction boundary, the US military has expressed concern it might have to shoot down a Russian aircraft over Syria if the Russian plane is seen as a threat to US or coalition forces fighting ISIS.
"The greatest concern is that we could shoot down a Russian aircraft because its actions are seen as a threat to our air or ground forces," Pickart told CNN.