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Genetics plays an increasing role in modern life as evidenced by the development of revolutionary techniques such as CRISPR-based genome editing and the rise of personalized genome services. However, genetics is difficult to learn; known issues include its abstract nature, different scales, and technical language. Pedigree analysis is a convergence of these concepts, requiring use of multiple symbolic scales and understanding the relationships and nature of alleles, genes, and chromosomes. To measure student understanding of these concepts, as well as support biology educational reform toward student-centered instruction, we developed a formative assessment to provide reliable and valid evidence of student understanding, learning, and misconceptions for pedigree analysis. Nine multiple choice items targeted to four learning objectives were developed in an iterative process with faculty and student input. We designed distractor answers to capture common student misconceptions and deployed a novel statistical technique to assess the congruence of distractor language with targeted misconceptions. Psychometric analysis showed the instrument provides valid and reliable data and has utility to measure normalized learning gains. Finally, we employed cross-tabulation and distractor progression to identify several stable misconceptions that can be targeted for instructional intervention.