Despite the existent gender parity in undergraduate biology degree attainment, gendered differences in outcomes are prevalent in introductory biology courses. Less is known about whether these disparities persist at the upper-division level, after most attrition is assumed to have occurred. Here, we report the consistent presence of gender equity gaps across 35 offerings (10 years) of a large-enrollment upper-division biology course at a research-intensive public university. Multilevel modeling showed that women's grades were lower than men's, regardless of prior GPA. These gender gaps were present even when controlling for students' race/ethnicity, socioeconomic status, first-generation college-going status, international status, and transfer status. Class size, gender representation in the classroom, and instructor gender did not significantly relate to course grades. Student questionnaires in a subset of offerings indicated gendered differences in course anxiety, science identity, and science self-efficacy, which correlated with grade outcomes. These results suggest that women experience differential outcomes in upper-division biology, which may negatively influence their persistence in STEM fields postgraduation. Our findings suggest that gender disparities are a systemic problem throughout the undergraduate biology degree and underscore the need for further examination and transformation of upper-division courses to support all students, even at late stages of their degrees.