Williams and older sibling Venus have been at the forefront of the women's game for the past two decades, but both are in their mid-30s and reaching the twilight of their illustrious careers.
Pliskova and her identical twin Kristyna have a considerable way to go before they can be compared to the Americans, but Thursday's surprise result has brought their story to the global spotlight.
On Saturday, Karolina will play in her first grand slam singles final, taking on Germany's Angelique Kerber -- who will end Serena Williams' record-equaling 186-week reign in the top spot when the new rankings are released Monday.
It is the first time Karolina has been past the third round of a major tournament, while Kristyna failed to get through the qualifying matches.
Ever since they first picked up a tennis racket aged four in the Czech Republic, the Pliskovas have been making a name for themselves with their strong serves. Now 24 years of age, they are both junior grand slam champions and have won three doubles titles together on the women's tour.
Karolina, who beat Serena 6-2 7-6 (7-5) at Flushing Meadows Thursday to follow up her earlier success over Venus, remembers watching the Williams duo with Kristyna when they were little.
"The first memory about the sisters was that they had this thing on their hair," she said after beating Croatian Ana Konjuh to reach her first grand slam semifinal. "I think it was Venus, and it somehow broke and it was everywhere on the court."
"So that's the first memory about them. And then obviously they are there for so many years and they have so many titles together. And especially Serena now. She's just one of the best players in the world, so it would be honor to play her."
The Williams sisters, a little over a year apart in age, never competed in junior tennis and mostly played each other while being coached by their father, Richard Williams.
Their success on the women's tour has been unprecedented: They were the first sisters ever to be ranked No. 1 and No. 2 at the same time and have won 14 grand slam doubles titles.
Although men's tennis has had its fair share of successful identical twin combinations -- from Tim and Tom Gullikson in the 1970s to Bob and Mike Bryan dominating men's doubles this century -- there haven't been many in the women's game.
The left-handed Kristyna is two minutes older and is ranked 122nd, while her sister cracked the top 10 last year.
Growing up, they were competitive in just about everything.
"We were always a little bit fighting, about everything actually," Karolina told the Australian Open website in January.
"But now it's better because we don't see each other that often. Before, when we were always together, we were fighting about everything, clothes and everything. But it improved a lot. Also with age, we are older now so it's getting a little bit better."
READ MORE: The 'age of Serena'
As for Serena, she had this to say when asked if she'd ever entertained the thought of an identical copy of herself competing on the WTA Tour.
"It would be a living hell," she told reporters after overcoming fifth-seeded Romanian Simona Halep in three sets in the quarterfinals at Flushing Meadows in New York Wednesday.
Thursday night's semifinal clash between the six-time US Open champion Williams and underdog Pliskova pitted the two most dominant servers in the women's game against each other.
"I haven't played against many big servers recently," Williams said before the game.
It was Pliskova's stellar serve that helped her gain the advantage, especially in the first set. There is no one on the women's tour with a serve more lethal than the six-foot one-inch (1.86-meter) Czech, who has fired off a staggering 439 aces this year. That's 119 more than Williams, who has played fewer matches.
Williams, who according to John McEnroe has "the greatest serve in the history of tennis," had hit 60 aces at the US Open, compared with 32 for Pliskova.
Although six-foot 11-inch Ivo Karlovic is the men's ace leader in New York with 120 from four matches played, Gael Monfils of France leads the remaining men's semifinalists with 49 aces.
When it comes to efficiency, Pliskova is also strong. Although Williams leads the Tour this season with 74.9% of first serve points won, the Czech is a close second with 74.6%.
Williams can struggle against big-serving opponents. She was outplayed by German Sabine Lisicki at Wimbledon in 2013 while Garbine Muguruza upset her in the finals of Roland Garros earlier this year.
Pliskova's serve had certainly caught Serena's attention prior to the game.
"She definitely gets some speed on it, but her placement is really, really nice," said the soon-to-be No. 2. "Hopefully I'll be able to read them and play okay."
The other women's semifinals between former world No. 1 Caroline Wozniacki
and Australian Open champion Kerber
was a completely different match, with Kerber easing to victory over her Danish opponent 6-4 6-3.
Both players are known for their retrieving skills and once played an epic 48-shot moonball rally in Indian Wells in 2013.
Before Thursday, Williams and Pliskova had played each other only once, with the top-ranked American winning a close two-set match in Stanford two years ago.
"I was a completely different player at that time, I have improved a lot," Pliskova said after her 6-2 6-2 win over Konjuh, perhaps with more foresight than even she herself knew.
Although Pliskova first reached the top 10 a year ago, before the US Open she was the only top-20 player to have never reached the quarters of a major.
After tuning up for the year's final slam with a win over Kerber in the final at Cincinnati last month, her big breakthrough came in a dramatic encounter against seven-time major winner Venus Williams in the fourth round at Flushing Meadows. Pliskova eventually prevailed in a third-set tie-break after both women squandered match points.
Speaking to on-court interviewer Pam Shriver after defeating Serena, the Czech said she didn't "believe" what she had accomplished before adding, "Actually I do believe it."
"I always have a chance to beat anyone if I am playing my game but this is something amazing. I'm really excited to be in the final, especially to beat a player like this."
Look out Venus and Serena, there's another sister act in town.