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Background: Hypoderma bovis and H. sinense (Diptera: Oestridae) mainly parasitise cattle and yaks. The two parasites are pathogenic and cause economic losses that result from reduced amounts of livestock products, including milk, meat, and skin. Genetic diversity and population genetic structure of H. bovis and H. sinense have not been evaluated, but could be used to inform appropriate strategies to control these parasites. Methods: We cloned and sequenced part of the mitochondrial cytochrome c oxidase subunit I (COI) gene from 60 H. bovis isolates and 52 H. sinense isolates from five locations in Qinghai Province, China, to identify polymorphisms, and infer their phylogenetic relationships, historical population expansions, and divergence time. Results: We identified 17 COI haplotypes from the H. bovis samples, and 23 COI haplotypes from the H. sinense samples. The haplotype and nucleotide diversities were 0.738 and 0.00202 for H. bovis, and 0.867 and 0.00300 for H. sinense, respectively, which indicates rich genetic diversity in H. bovis and H. sinense populations. Bayesian phylogenetic analysis revealed that the two species are monophyletic, and geographical structuring of haplotypes was significantly different in H. sinense (P < 0.05), but not H. bovis. Neutrality tests and mismatch distribution statistical analysis revealed that populations of the two species have undergone demographic expansions. The divergence three Hypoderma spp. (H. bovis, H. lineatum, and H. sinense) was estimated to have occurred approximately 4.5 million years ago (Mya), which indicates that the rapid uplift of the Qinghai-Tibetan Plateau during the late Miocene-Pliocene was associated with divergence of Hypoderma species. Conclusions: Results of the present study revealed that both H. bovis and H. sinense displayed high genetic diversity and widespread population genetic differentiation within and among populations; these data, along with the molecular phylogeny, demographic history, and divergence time estimation, provide new insight into evolutionary history of these species. These findings will help elucidate speciation in Hypoderma and provide theoretical basis for epidemiological surveillance and control of these species on the Qinghai-Tibetan Plateau. [ABSTRACT FROM AUTHOR]