Understanding sources of performance bias in science assessment provides important insights into whether science curricula and/or assessments are valid representations of student abilities. Research investigating assessment bias due to factors such as instrument structure, participant characteristics, and item types are well documented across a variety of disciplines. However, the relationships among these factors are unclear for tasks evaluating understanding through performance on scientific practices, such as explanation. Using item-response theory (Rasch analysis), we evaluated differences in performance by gender on a constructed-response (CR) assessment about natural selection (ACORNS). Three isomorphic item strands of the instrument were administered to a sample of undergraduate biology majors and nonmajors (Group 1: n = 662 [female = 51.6%]; G2: n = 184 [female = 55.9%]; G3: n = 642 [female = 55.1%]). Overall, our results identify relationships between item features and performance by gender; however, the effect is small in the majority of cases, suggesting that males and females tend to incorporate similar concepts into their CR explanations. These results highlight the importance of examining gender effects on performance in written assessment tasks in biology.