We have sequenced the genome of a secondDrosophilaspecies,Drosophila pseudoobscura, and compared this to the genome sequence ofDrosophila melanogaster, a primary model organism. Throughout evolution the vast majority ofDrosophilagenes have remained on the same chromosome arm, but within each arm gene order has been extensively reshuffled, leading to a minimum of 921 syntenic blocks shared between the species. A repetitive sequence is found in theD. pseudoobscuragenome at many junctions between adjacent syntenic blocks. Analysis of this novel repetitive element family suggests that recombination between offset elements may have given rise to many paracentric inversions, thereby contributing to the shuffling of gene order in theD. pseudoobscuralineage. Based on sequence similarity and synteny, 10,516 putative orthologs have been identified as a core gene set conserved over 25–55 million years (Myr) since thepseudoobscura/melanogasterdivergence. Genes expressed in the testes had higher amino acid sequence divergence than the genome-wide average, consistent with the rapid evolution of sex-specific proteins.Cis-regulatory sequences are more conserved than random and nearby sequences between the species—but the difference is slight, suggesting that the evolution ofcis-regulatory elements is flexible. Overall, a pattern of repeat-mediated chromosomal rearrangement, and high coadaptation of both male genes andcis-regulatory sequences emerges as important themes of genome divergence between these species ofDrosophila.