Introduction: homosexuality and transsexuality are mediated by biological mechanisms of genetic type, early action of hormones and by environmental mechanisms, affecting structural and functional changes in various brain regions. The aim of this research is describe the biological knowledge of homosexuality and transsexuality for students of Physical Education. Method: an ad hoc questionnaire was built and applied to 305 students of Pedagogy in Physical Education from three universities in Chile. Results: the questionnaire was constituted by 18 items, in addition it presents six factors that explain the 58.378% of variance and a Cronbach alpha of .763. The item that presented the most successful answers was There is a "gay gene" that determines homosexuality and lesbianism and the one that presents the least number of successful was When a woman is a lesbian there is more chance that her sister is also a lesbian. Conclusion: Analyses show that the instrument is valid and reliable for assessing the biological knowledge of homosexuality and transsexuality in students of Physical Education Pedagogy. In 14 items more than 50% of the sample answered I do not know and 78.6% of the sample obtained only between one and six correct answers.