We analyzed a 5,770-bp genomic region of Drosophila virilis that contains a cluster of two maltase genes showing sequence similarity with genes in a cluster of three maltase genes previously identified in Drosophila melanogaster. The D. virilis maltase genes are designated Mav1 and Mav2. In addition to being different in gene number, the cluster of genes in D. virilis differs dramatically in intron-exon structure from the maltase genes in D. melanogaster, the transcriptional orientation of the genes in the cluster also differs between the species. Our findings support a model in which the maltase gene cluster in D. virilis and D. melanogaster evolved independently. Furthermore, while in D. melanogaster the maltase gene cluster lies only 10 kb distant from the larval cuticle gene cluster, the maltase and larval cuticle gene clusters in D. virilis are located very far apart and on a different chromosome than that expected from the known chromosome arm homologies between D. virilis and D. melanogaster. A region of the genome containing the maltase and larval cuticle gene clusters appears to have been relocated between nonhomologous chromosomes.