Introduction: Various studies relate variables such as age, sex, socioeconomic level, religion, etc. with levels of homophobia and transphobia. The aim of this research is to determine the incidence of some sociodemographic variables and biological knowledge about homosexuality and transsexuality on the levels of homophobia and transphobia in physical education students in Chile. Method: a sociodemographic survey was applied, the scale of homophobia EHF-10, the scale of negative attitudes towards trans people and the questionnaire of biological knowledge of homosexuality and transsexuality to 463 students of Pedagogy in Physical Education from six universities in Chile. Results:The levels of homophobia and transphobia are low in the sample, being the coexistence with friends/ or known/homosexual, bisexual and trans, and the religious beliefs the factors that most affect those negative attitudes. A negative and low relationship was observed between biological knowledge of homosexuality and transsexuality and levels of homophobia and transphobia. Conclusion:The sample has low levels of homophobia and transphobia.There are also low biological knowledge values of homosexuality and transsexuality, with a low and negative relationship between such knowledge and negative attitudes towards homosexuals and transgender people.