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A brief glance through molecular biology and biochemistry textbooks underscores the importance of interpreting visual images in the molecular life sciences. In fact, biomolecular visual literacy has been deemed a threshold concept, essential for student success in the field. As one example, grasping the information displayed in visual representations is a gateway to a deep understanding of structure-function relationships, a core concept in biology education. Despite much interest, few studies have examined the assessment of visual literacy skills in the area of biomolecules. Ten years ago, BioMolViz began an initiative to improve biomolecular visualization instruction and assessment, which focused on developing validated assessments to probe students' visual literacy skills. In 2023, we introduced the BioMolViz Library, a repository where instructors can access the instruments built by our community. A subset of these assessments were administered in classrooms in a pilot field test during the 2022–2023 academic year. We gained invaluable information from both quantitative and qualitative data collected. Lessons learned from this first classroom test guided the design of the 2023–2024 large-scale field testing we describe here with over ten partner institutions, high enrollment classes, and an increased number of items per survey. We present the results of our analysis of item difficulty, discrimination, and distractor analysis, alongside a robust analysis of the influence of gender and race/ethnicity on student performance. To improve the statistical power of the study, we exchanged open-ended written feedback for an increased number of assessment items administered on each survey. However, recognizing the value of student feedback obtained through a mixed methods analysis from our 2022–2023 study, we followed up with focus groups to explore the perceptions and problem solving process of both low- and high-performing students. We present the results of our assessment validation, including an analysis of the influence of learner level, gender identity, and race/ethnicity on performance. We include suggestions for equitable and inclusive assessment methods as we continue to strive to improve visual literacy instruction. This material is supported by the National Science Foundation (NSF) under grants RCN-UBE #1920270 and NSF-IUSE #1712268.