AbstractMeiotic crossing over ensures proper segregation of homologous chromosomes and generates genotypic diversity. Despite these functions, little is known about the genetic factors and population genetic forces involved in the evolution of recombination rate differences among species. The dicistronic meiosis gene, mei-217/mei-218, mediates most of the species differences in crossover rate and patterning during female meiosis between the closely related fruitfly species, Drosophila melanogaster and D. mauritiana. The MEI-218 protein is one of several meiosis-specific mini-chromosome maintenance (mei-MCM) proteins that form a multi-protein complex essential to crossover formation, whereas the BLM helicase acts as an anti-crossover protein. Here we study the molecular evolution of five genes— mei-218, the other three known members of the mei-MCM complex, and Blm— over the phylogenies of three Drosophila species groups— melanogaster, obscura, and virilis. We then use transgenic assays in D. melanogaster to test if molecular evolution at mei-218 has functional consequences for crossing over using alleles from the distantly related species D. pseudoobscura and D. virilis. Our molecular evolutionary analyses reveal recurrent positive selection at two mei-MCM genes. Our transgenic assays show that sequence divergence among mei-218 alleles from D. melanogaster, D. pseudoobscura, and D. virilis has functional consequences for crossing over. In a D. melanogaster genetic background, the D. pseudoobscura mei-218 allele nearly rescues wildtype crossover rates but alters crossover patterning, whereas the D. virilis mei-218 allele conversely rescues wildtype crossover patterning but not crossover rates. These experiments demonstrate functional divergence at mei-218 and suggest that crossover rate and patterning are separable functions.