Teaching practices used by teachers influence students' achievement of learning outcomes. These practices may be explored from students' perspectives. Because gender may influence students' perceptions of learning environments, this study explored gender differences in students' perceptions of teaching practices employed in their biology classrooms. The study employed a quantitative survey design with a sample of 469 students (223 females, 246 males) from eight secondary schools purposively selected from four districts in Zambia. The data were collected using a Likert-scale questionnaire consisting of 27 items categorised into four domains of teaching practices as enacted Pedagogical Content Knowledge: students' prior knowledge, curriculum interpretation, instructional strategies and representations and assessment. The questionnaire had a reliability index of 0.842. Data were analysed using the Statistical Package for the Social Sciences by computing frequencies (percentages) and chi-square tests for gender differences for each item. The findings revealed significant gender differences in students' perceptions for nine teaching practices, with more males than females reporting agreement with the use of particular teaching practices. Significantly more males are satisfied with the teacher's strategies focusing on their personal learning needs, whereas significant more female students appreciate their teacher's highlighting of common misconceptions. Significantly more male students appreciate instructional strategies such as practical work and storytelling, whereas significantly more female students appreciate the use of graphs, diagrams, etc. Significantly more male students appreciate the variety in their teacher's question technique and assessment formats. The results suggest the need to use gender-inclusive practices in biology lessons. The study recommends improving teachers' teaching practices reflecting gender differences, learner-centredness and diversity in assessment through professional development activities. Furthermore, future research may explore ways of reducing gender differences, i.e. practices reducing female perceived disadvantage.