One proposal for the persistence of homosexuality in the human population is the sexually antagonistic gene hypothesis, which suggests that the lower fertility of homosexual individuals, especially men, may be compensated by higher fertility of their relatives of the opposite sex. To test this hypothesis, we have collected data from 7,312 heterosexual men, 459 gay men, 3,352 heterosexual women, and 79 lesbian women mainly from Czechia. In an online survey, participants answered questions regarding their own as well as their parents’ and grandparents’ fertility. For men, we obtained no significant results except for higher fertility of gay men’s paternal grandmothers, but the magnitude of this effect was very small. For the female sample, we recorded lower fertility of lesbian women’s mothers and fathers. In line with our expectations, both gay men and lesbian women had lower fertility rates than their heterosexual counterparts. Our results are consistent with recent studies which likewise do not support the sexually antagonistic gene hypothesis.