Diptera (true flies) are among the most diverse holometabolan insect orders and were the first eukaryotic order to have a representative genome fully sequenced. 110 fly species have publically available genome assemblies and many hundreds of population-level genomes have been generated in the model organisms Drosophila melanogaster and the malaria mosquito Anopheles gambiae. Comparative genomics carried out in a phylogenetic context is illuminating many aspects of fly biology, providing unprecedented insight into variability in genome structure, gene content, genetic mechanisms, and rates and patterns of evolution in genes, populations, and species. Despite the rich availability of genomic resources in flies, there remain many fly lineages to which new genome sequencing efforts should be directed. Such efforts would be most valuable in fly families or clades that exhibit multiple origins of key fly behaviors such as blood feeding, phytophagy, parasitism, pollination, and mycophagy.