The concern with the well-being of users in buildings has become increasingly essential, covering aspects related to health, energy efficiency, and productivity. The thermal environment evaluation in buildings has become more frequent due to the time people spend inside them. In this context, this study aimed to analyze thermal comfort in classrooms at a Brazilian University. During the autumn, 50 measurements were performed, resulting in 519 valid responses. The results of the linear regression analysis revealed that the thermal comfort range for females was 20.39–22.19 °C, while for males it was 19.47–22.56 °C. Through discriminant analysis, participants were classified based on their thermal sensation vote (TSV), predicted mean vote (PMV), and thermal preference votes (PREF), achieving a success rate of 76.1% for females and 81.6% for males in forming the groups, which demonstrates the effectiveness of discriminant functions in predicting thermal comfort for both groups. These results highlight the importance of considering gender differences in the search for thermal comfort conditions and providing guidelines that promote the well-being of occupants and the conscious use of energy. This implies adjusting the thermal conditions according to the specific needs of males and females in classrooms, always seeking to provide a suitable environment for activities, and considering energy efficiency and users’ productivity.