AbstractIn this article, we describe an inexpensive, two‐session undergraduate laboratory activity that introduces important molecular biology methods in the context of biodiversity. In the first session, students bring tentatively identified flies (order Diptera, true flies) to the laboratory, extract DNA, and amplify a region of the mitochondrial gene NADH dehydrogenase subunit 1. In the second session, the students digest the PCR product with a restriction enzyme, visualize the resulting fragments by agarose gel electrophoresis, and analyze their results with comparison to known sequences. The diversity of flies and their importance as disease vectors, agriculture pests, pollinators, models of speciation, and in the case of Drosophila melanogaster, as a genetic model organism, offer many perspectives with which to appeal to students' interests. The laboratory exercise can be linked as a module to topics in biodiversity, bioinformatics, entomology, evolution, and mutagenesis.