Video games have become a popular medium in our society, and recent scholarship suggests that games can support substantial learning. This study stems from a project in which we created a video game enabling students to use biotechnology to solve a societal problem. As students engaged in the game, they necessarily interacted with the underlying biological principles. The study focused on the extent to which students learned biology concepts in the context of a game-based curriculum. In particular, we explored learning among high school students, stratified across different academic levels (i.e., general, honors, and advanced). We used a pre/post-test repeated measures multiple analysis of variance (MANOVA) design and a multi-level assessment strategy with assessments positioned at variable distances from the curriculum to illuminate the nature of student learning and the potential interactions of academic level and learning associated with the game-based curriculum. A total of 647 students from 31 different biology classes, taught by 10 different teachers, participated in the study. Results indicated statistically and practically significant gains in student performance on both a proximal (curriculum-aligned) test and a distal (standards-aligned) exam of biological content knowledge. Students from classes across all three academic levels demonstrated gains with relatively large effect sizes on the proximal test. Whereas the honors and advanced students demonstrated less substantial gains on the distal exam, the general students demonstrated gains of similar effect size on both the proximal and distal assessments. These results suggest that, despite teacher concerns about the potential effectiveness of gaming to support learning among students in lower academic levels, game-based curricula can support learning of important biological content for students of varying academic levels and may, in fact, be most beneficial for lower level students. The affordances of employing a multi-level assessment model for the investigation of innovative curricula are discussed. (Contains 3 figures and 5 tables.)