The conserved relationship between orthologs of many cytochrome P450 genes involved in ecdysone synthesis is not reflected in the evolution of the Drosophila Cyp307a genes. In Drosophila melanogaster Cyp307a1 (spook) and Cyp307a2 (spookier) both play essential roles in ecdysone synthesis and may possess biochemically redundant functions. Using phylogenetic analyses we show that the Drosophila Cyp307a genes were formed from two independent duplication events depicting a complicated evolutionary scenario. An initial duplication, from a Cyp307a2 ancestral gene produced the Cyp307a1 gene that has been maintained only in the Sophophoran subgenus. A second duplication in the Drosophila subgenus formed an additional paralog, Cyp307a3. Microsynteny is conserved for Cyp307a2 throughout the Drosophila species, but is not conserved between Cyp307a1 and Cyp307a3. These are located in different genomic positions in the Sophophora and Drosophila subgenera, respectively. Cyp307a3 appears to encode a functional gene product and is expressed in a different spatial and temporal manner to Cyp307a1. This suggests some level of functional divergence between the Cyp307a paralogs in different Drosophila species.