Evolution by natural selection is the fundamental backbone of the life sciences. Therefore, it is important for teacher education programs to ensure graduates possess a strong knowledge of evolution in order to teach at all levels of biology education. The main aim was to investigate the impacts of the Slovene pre-service preschool and primary school teachers’ religiosity and educational background on their evolutionary content knowledge. In the present study, understanding of five evolutionary topics, religiosity and educational background of 269 students was studied. Results show that students have a very poor understanding of evolution. They very often use teleological reasoning. Although many pieces of research have shown that religiosity can be in conflict with evolutionary theory, our findings show that religiosity does not significantly correlate with evolutionary knowledge nor to the educational background of students. However, for students’ understanding of evolution, it is important how many years of biology lessons they had in secondary school. This should be better taken into account by educational policymakers because evolutionary principles are becoming increasingly relevant in medicine, agriculture and other socio-scientific topics.