Promoting students' ability to engage in discipline-specific practices is a central goal of chemistry education. Yet if instruction is to meaningfully foster such ability, we must first understand students' reasoning during these practices. By characterizing constraints on chemistry students' reasoning, we can design instruction that targets this constrained reasoning and ultimately promotes more sophisticated ways of thinking. For this study, we investigated reasoning used by 18 organic chemistry students at a large university in the United States as they evaluated the success of chemical syntheses through IR and [superscript 1]H NMR spectral interpretation, a common task of practicing chemists. Students completed a series of interpretation tasks while having their eye movements tracked and then participated in semi-structured, cued retrospective think-aloud (RTA) interviews about their reasoning during spectral interpretation. RTA interviews were analyzed qualitatively to characterize invalid chemical assumptions and heuristic reasoning strategies used by participants, both of which science education literature identifies as fundamental constraints to learning. The most problematic assumptions and heuristics, i.e., those used more frequently by unsuccessful participants, were then identified through statistical analysis. Findings suggest that the most problematic constraints on students' reasoning during spectral interpretation constitute a combination of particular invalid chemical assumptions and heuristic reasoning strategies.