The WTA said the once-in-a-lifetime test for the SRY gene, which helps in determining biological sex, can be conducted via a cheek swab or blood test, with the new policy coming into effect on Tuesday.
The previous WTA policy permitted transgender women to participate if they declared their gender as female and then maintained a lowered testosterone level for a period of two years before competing.
There are no known transgender women currently active at any level of professional tennis.
"Eligibility to participate in WTA tournaments will be confirmed through a required, one-time screening process for the SRY gene," a WTA spokesperson told Reuters.
"Player screening will begin in 2026, following all appropriate confidentiality and data privacy safeguards and concurrent with a comprehensive player engagement program.
"The WTA recognises that this is a sensitive and complex issue and is committed to treating all players with dignity and implementing the policy in a respectful and thoughtful way."
Transgender player Renee Richards competed on the women's professional tour from 1977 to 1981 before coaching tennis great and gay rights trailblazer Martina Navratilova.
Navratilova, an 18-time Grand Slam singles champion, has been an outspoken critic of the inclusion of transgender athletes, while world No.1 Aryna Sabalenka last year said it would be unfair for women to face transgender players.
Others, such as Billie Jean King — the 12-time Grand Slam singles champion who won the original "battle of the sexes" in 1973 — see transgender exclusion as discrimination.
Several sports federations, including World Athletics and World Boxing, have introduced similar gene tests in recent years.
In March, the International Olympic Committee said only biological female athletes whose gender has been determined by a one-time test would be eligible to take part in female category events at the Olympic Games.
Transgender advocacy groups say excluding transgender athletes amounts to discrimination.
Critics of transgender inclusion say going through male puberty imbues athletes with a musculo-skeletal advantage that transition does not mitigate.
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