Bot flies (Oestridae) are obligate endoparasites of mammals, and their extraordinary diversification is of great importance in understanding the evolution of parasitism. However, evolutionary analysis of Oestridae has long been impeded by lack of information. Here, the first three mitochondrial genomes of nasal bot flies (Cephalopina titillator, Cephenemyia trompe and Rhinoestrus usbekistanicus) and a comparative mitochondria! genomic analysis between subfamilies of Oestridae arc presented. Contrasting to many other parasites, mitochondria! genomes of oestrids are conserved in structure, and genes have retained the same order and direction as the ancestral insect mitochondria) genome. Nucleotide composition is highly heterogenous, with Gasterophilinae possessing highest GC content and smallest genomic size. Mitochond rial evolutionary rates vary considerably, with Hypodermatinae and Oestrinae exhibiting a faster average rate than Cuterebrinae and Gasterophilinae. In addition, the first phylogenomic analysis covering all four bot fly subfamilies was conducted, supporting monophyly of Oestridae and a sister-group relationship of Hypodermatinae and Oestrinae. The only topological ambiguity is Cuterebrinae being a sister-group of either (Hypodermatinae + Oestridae) or Gasterophilinae. Thus, we suggest that mitochondria! genomes carry a great potential for phylogenetic analysis of Oestridae, and more information of Cuterebrinae is needed to illuminate the early evolutionary radiation and parasite-host coevolution of bot flies. (C) 2020 Published by Elsevier B.V.