Novotna, who beat France’s Nathalie Tauziat to win Wimbledon in 1998, had been battling cancer for several years.
A former world number one in doubles and number two in singles, Novotna had celebrated her 49th birthday last month.
But the WTA announced in a statement: “It is with deep sadness that the WTA announces the passing on Sunday, November 19, of Jana Novotna, the former WTA World No.1 doubles and No.2 singles champion. After a long battle with cancer, Jana died peacefully, surrounded by her family in her native Czech Republic, aged 49.”
“Inducted into the International Tennis Hall of Fame in 2005, her career successes made Jana Novotna an inspiration to those who knew her and to many up and coming WTA players on the professional women’s tennis circuit. In more recent years, she made the transition into professional coaching.”
Steve Simon, WTA CEO, added: “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.”
The Czech player lost in the Wimbledon final in 1993 and 1997 but finally won the coveted trophy in 1998 when he defeated Frenchwoman Tauziat.
In total, Novotna won 24 WTA singles titles and 76 doubles trophies in 14 years on the professional tour.
American former tennis player Pam Shriver tweeted: “Jana was as kind as she was athletic, as smart as she was competitive.
“I can’t believe she is gone this soon. Her smile lives forever young.”
Ex-British player Andrew Castle, now a commentator for the BBC, said: “What a terrible shame. She was good fun of an evening, a champion on court, so tough to play against. And her smile was wonderful.”
Former Czech tennis player Iveta Benesova added: “R.I.P. Jana Novotna. Will always be my childhood hero! Leaving too soon, so sad… #speechless.”
Novotna, whose serve and volley playing style won plenty of admirers, was inducted into the International Tennis Hall of Fame in 2015.
She won over 500 career singles matches but it was her accomplishments in doubles that earned her most plaudits, winning 12 doubles and four mixed doubles titles across all four Grand Slams.
Altogether, Novotna won 17 Grand Slam titles.
Novotna was 6-7, 6-1, 4-1 up against Germany’s Graf, who had been favourite for the title.
But the Czech could’t dispatch a break point that would have put her a game away from the championship, and Graf came back in emphatic fashion.
The endearing images of the Duchess of Kent consoling a tearful Novotna on Centre Court remain to this day.