Undergraduate students enrol at university with diverse levels of prior knowledge and intuitive conceptions about essential biological topics. This longitudinal study explored the development of a conceptual understanding about three topics and how domain-specific prior knowledge affects this development. Undergraduate students (N = 50) of biological and environmental sciences participated in three measurement points – baseline (2019), follow-up 1 (2020), and follow-up 2 (2021) – where the same questionnaire with open-ended tasks was given. A mixed-methods approach was used for quantitative scoring of the answers and qualitative thematic analysis to describe the development of individual students’ conceptual understanding. The effect of prior knowledge was studied by dividing the students into two groups based on the baseline performance. The topic of evolution showed the most significant differences in prior knowledge and in knowledge gains. Students with lower prior knowledge experienced desirable shifts in their explanatory models about evolutionary processes, but an equal amount exhibited fragmented learning trajectories. Students with a more robust prior understanding of such an emergent phenomenon can more likely integrate scientific concepts in their knowledge framework. Implications for teaching in higher education are discussed.