The Drosophila 12 Genomes Consortium analyzed the genomes of 12 Drosophila species, including 10 newly sequenced ones, to improve evolutionary inference. These genomes reveal how sequence divergence across species can illuminate evolutionary processes on a genomic scale. The data enhance Drosophila's genetic tools, aiding research in development, cell biology, genetics, disease, neurobiology, behavior, physiology, and evolution. Despite similarities, many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions were identified, possibly underlying ecological and behavioral differences. Drosophila species vary in morphology, ecology, and behavior, spanning diverse habitats. The 12 sequenced species originate from Africa, Asia, the Americas, and the Pacific Islands, including cosmopolitan species like D. melanogaster and D. simulans. The genomes provide an unprecedented dataset to contrast genome structure, content, and evolutionary dynamics across the Drosophila phylogeny. Genome sequencing and assembly involved whole-genome shotgun sequencing for 10 species, with varying coverage. Assembly methods improved genome quality, and comparative syntenic information helped refine assemblies. Mitochondrial and Wolbachia genomes were also sequenced. Repeat and transposable element annotation used multiple methods to estimate content. Protein-coding gene annotation combined de novo and homology-based predictors, with GLEAN used to combine predictions. Homology assignments used BLAST-based methods and synteny-aided approaches. Gene models were validated using expression data, with many showing expression. Transposable element-contaminated gene models were excluded. Homology calls were reconciled using FRB and Synpipe methods. Gene family evolution showed expansions and contractions, with some families evolving rapidly. Lineage-specific genes were identified, with unique characteristics. Protein-coding gene evolution showed positive selection and selective constraints, with some genes evolving more slowly. Factors like gene expression, essentiality, and chromosomal location influenced protein evolution rates. Chromosomal context modulated evolutionary rates, with the X chromosome showing elevated codon bias and positive selection. These findings highlight the dynamic nature of genome structure and evolution in Drosophila.The Drosophila 12 Genomes Consortium analyzed the genomes of 12 Drosophila species, including 10 newly sequenced ones, to improve evolutionary inference. These genomes reveal how sequence divergence across species can illuminate evolutionary processes on a genomic scale. The data enhance Drosophila's genetic tools, aiding research in development, cell biology, genetics, disease, neurobiology, behavior, physiology, and evolution. Despite similarities, many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions were identified, possibly underlying ecological and behavioral differences. Drosophila species vary in morphology, ecology, and behavior, spanning diverse habitats. The 12 sequenced species originate from Africa, Asia, the Americas, and the Pacific Islands, including cosmopolitan species like D. melanogaster and D. simulans. The genomes provide an unprecedented dataset to contrast genome structure, content, and evolutionary dynamics across the Drosophila phylogeny. Genome sequencing and assembly involved whole-genome shotgun sequencing for 10 species, with varying coverage. Assembly methods improved genome quality, and comparative syntenic information helped refine assemblies. Mitochondrial and Wolbachia genomes were also sequenced. Repeat and transposable element annotation used multiple methods to estimate content. Protein-coding gene annotation combined de novo and homology-based predictors, with GLEAN used to combine predictions. Homology assignments used BLAST-based methods and synteny-aided approaches. Gene models were validated using expression data, with many showing expression. Transposable element-contaminated gene models were excluded. Homology calls were reconciled using FRB and Synpipe methods. Gene family evolution showed expansions and contractions, with some families evolving rapidly. Lineage-specific genes were identified, with unique characteristics. Protein-coding gene evolution showed positive selection and selective constraints, with some genes evolving more slowly. Factors like gene expression, essentiality, and chromosomal location influenced protein evolution rates. Chromosomal context modulated evolutionary rates, with the X chromosome showing elevated codon bias and positive selection. These findings highlight the dynamic nature of genome structure and evolution in Drosophila.