Primary literature offers rich opportunities to teach students how to "think like a scientist," but the challenges students face when they attempt to read research articles are not well understood. Here, we present an analysis of what master's students perceive as the most challenging aspects of engaging with primary literature. We examined 69 pairs of pre- and postcourse responses from students enrolled in a master's-level course that offered a structured analysis of primary literature. On the basis of these responses, we identified six categories of challenges. Before instruction, "techniques" and "experimental data" were the most frequently identified categories of challenges. The majority of difficulties students perceived in the primary literature corresponded to Bloom's lower-order cognitive skills. After instruction, "conclusions" were identified as the most difficult aspect of primary literature, and the frequency of challenges that corresponded to higher-order cognitive skills increased significantly among students who reported less experience with primary literature. These changes are consistent with a more competent perception of the primary literature, in which these students increasingly focus on challenges requiring critical thinking. Students' difficulties identified here can inform the design of instructional approaches aimed to teach students how to critically read scientific papers.