Visual literacy, which is the ability to effectively identify, interpret, evaluate, use, and create images and visual media, is an important aspect of science literacy. As molecular processes are not directly observable, researchers and educators rely on visual representations (e.g., drawings) to communicate ideas in biology. How learners interpret and organize those numerous diagrams is related to their underlying knowledge about biology and their skills in visual literacy. Furthermore, it is not always obvious how and why learners interpret diagrams in the way they do (especially if their interpretations are unexpected), as it is not possible to "see" inside the minds of learners and directly observe the inner workings of their brains. Hence, tools that allow for the investigation of visual literacy are needed. Here, we present a novel card-sorting task based on visual literacy skills to investigate how learners interpret and think about DNA-based concepts. We quantified differences in performance between groups of varying expertise and in pre- and postcourse settings using percentages of expected card pairings and edit distance to a perfect sort. Overall, we found that biology experts organized the visual representations based on deep conceptual features, while biology learners (novices) more often organized based on surface features, such as color and style. We also found that students performed better on the task after a course in which molecular biology concepts were taught, suggesting the activity is a useful and valid tool for measuring knowledge. We have provided the cards to the community for use as a classroom activity, as an assessment instrument, and/or as a useful research tool to probe student ideas about molecular biology.