In a statement on its website, the WTA said the former world No.2 died peacefully "surrounded by her family in her native Czech Republic."
Novotna captured the hearts of fans when she burst into tears after losing to Steffi Graf in the 1993 Wimbledon final and was consoled by the Duchess of Kent. It continues to be one of the most memorable moments in the tournament's history.
She also lost the 1997 Wimbledon final before going on to win the title in 1998, beating Nathalie Tauziat in the final.
Tennis great Martina Navratilova described Novotna as a "true friend" and an "amazing woman."
"The tennis world is so sad about the passing of Jana Novotna... I am gutted and beyond words," tweeted the Czechoslovakia-born Navratilova, a former world No.1 and winner of 18 singles Grand Slam titles.
Two-time Wimbledon champion and compatriot Petra Kvitova said Novotna had inspired her and supported her during her career.
The world No.29 posted on Instagram: "Today we lost a wonderful person, a Czech who played beautiful tennis, who inspired me and supported me.
"I remember our hugs and our tears after my Wimbledon titles. Thank you Jana. We will miss you."
During an illustrious career, Novotna won 17 Grand Slam titles, including 12 in doubles and four in mixed doubles, and was a member of the Czech Republic's victorious 1998 Fed Cup team.
She also won two Olympic silver medals in doubles, in 1988 and 1996, and a singles bronze at the 1996 Atlanta Games.
Novotna, renowned for her serve and volley, was inducted into the Tennis Hall of Fame in 2005.
Steve Simon, chief executive of the WTA, said: "Jana was an inspiration both on and off court to anyone who had the opportunity to know her. Her star will always shine brightly in the history of the WTA. Our condolences and our thoughts are with Jana's family."
'I believe you will do it'
During her first Wimbledon final, Novotna, then aged 24, had led 4-1 in the deciding set and seemed on course to cause an upset against the great Graf only to fall to a 7-6 (8-6) 1-6 6-4 defeat after losing five games in a row.
When presented with the runner's-up trophy, Novotna started to cry and the Duchess of Kent comforted her on Centre Court in a touching moment.
Novotna said the Duchess had told her "I believe you will do it" and, indeed, she did, winning Wimbledon, aged 29 and nine months, to become the oldest first-time Grand Slam singles winner in the Open era.
Tributes to Novotna
Former world No.1 and 18-time Grand Slam champion Chris Evert was also among those to pay tribute to Novotna on social media.
The American described Novotna as a "woman with integrity and honor."
Czech Lucie Safarova, the current world No.30, posted on Instagram, while Wimbledon's official Twitter account also paid tribute to the tennis star.