Researchers have called for undergraduate courses to update teaching frameworks based on the Modern Synthesis with insights from molecular biology, by stressing the molecular underpinnings of variation and adaptation. To support this goal, we developed a modified version of the widely used Assessing Conceptual Reasoning of Natural Selection (ACORNS) instrument. The expanded tool, called the E-ACORNS, is explicitly designed to test student understanding of the connections among genotypes, phenotypes, and fitness. E-ACORNS comprises a slight modification to the ACORNS open-response prompts and a new scoring rubric. The rubric is based on five core concepts in evolution by natural selection, with each concept broken into elements at the novice, intermediate, and expert-level understanding. Initial tests of the E-ACORNS showed that (1) upper-level undergraduates can score responses reliably and quickly, and (2) students who were just starting an introductory biology series for majors do not yet grasp the molecular basis of phenotypic variation and its connection to fitness.