Threshold concepts (TCs) are concepts that, when mastered, represent a transformed understanding of a discipline without which the learner cannot progress. We have undertaken a process involving more than 75 faculty members and 50 undergraduate students to identify a working list of TCs for biochemistry. The process of identifying TCs for biochemistry was modeled on extensive work related to TCs across a range of disciplines and included faculty workshops and student interviews. Using an iterative process, we prioritized five concepts on which to focus future development of instructional materials. Broadly defined, the concepts are steady state, biochemical pathway dynamics and regulation, the physical basis of interactions, thermodynamics of macromolecular structure formation, and free energy. The working list presented here is not intended to be exhaustive, but rather is meant to identify a subset of TCs for biochemistry for which instructional and assessment tools for undergraduate biochemistry will be developed.