The current study investigates pre-service teachers’ knowledge and misconceptions of learning with multimedia (i. e., text and pictures) and whether they are dependent on the pre-service teachers’ subject combination and study progress. To this end, 88 student teachers from the University of Education answered both yes-no questions about typical misconceptions (“Both hemispheres are activated when learning with text and pictures, whereas only one hemisphere is activated when learning with just text”; “learning with text and pictures is always more effective than learning with text alone”), and scenario-based questions about the functions of pictures when they are presented in combination with text. The results suggest that pre-service teachers have some misconceptions and specific knowledge gaps about the cognitive functions of pictures, regardless of their subject combination and study progress. Further research should aim at testing interventions on how to both close these knowledge gaps and specifically address these misconceptions.