This study aims to investigate pre-service science teachers' knowledge levels, practical experiences, and perceived competencies in teaching biodiversity at secondary school level. The study was conducted in Turkey with 151 pre-service science teachers, 50 of whom were first-year, 34 second year, 42 third year, and 25 fourth year during the fall semester of 2022-2023. An open-ended biodiversity concept test was applied to identify their knowledge levels, prior education experiences, and perceived competencies in teaching biodiversity. Descriptive statistics and content analysis results revealed that as the year level increased, the knowledge levels and experiences of the pre-service teachers increased as well, and in parallel, they perceived themselves as more competent in teaching the subject of biodiversity. When first and second year students were evaluated together, it was found that most of their answers were at a low level in terms of their knowledge about biodiversity. When the third and fourth grade teacher candidates' knowledge levels about biodiversity were examined, it was revealed that they were predominantly at a good level. As can be clearly seen, as year levels increased, the level of knowledge also increased. These results highlight the importance of theoretical and practical courses for content and pedagogical knowledge on the perceived competencies of the pre-service teachers in teaching biodiversity.