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Background: Gasterophilus species are widely distributed around the world. The larvae of these flies parasitize the digestive tract of equids and cause damage, hindering horse breeding and protection of endangered species. However, study of the genetic structure of geographically distinct Gasterophilus populations is lacking. Here, we analyzed the genetic diversity of Gasterophilus pecorum, G. intestinalis, G. nasalis and G. nigricornis from three typical grasslands (meadow, desert and alpine steppes) in China as compared to published sequences from Italy, Poland and China (Daqing and Yili), based on the mitochondrial cytochrome c oxidase cox1 and cox2 gene sequences. Results: Haplotype diversity and nucleotide diversity of mitochondrial genes was generally high in all Gasterophilus populations. Due to the unique natural climatic conditions of the alpine steppe, there were high levels of genetic differentiation among different geographical populations of G. pecorum and G. nasalis, indicating that environmental variations influenced population genetic structure. Frequent exchanges between meadow and desert steppe Gasterophilus species resulted in low genetic differentiation. The highest exchange rates were found among G. intestinalis populations. Genetic differentiation was only observed on a large geographical scale, which was confirmed by analyzing population genetic structure. Three species, G. pecorum, G. intestinalis and G. nasalis, from meadow steppe showed a high emigration rate, indicating that the direction of Gasterophilus dispersal in China was from east to west. Conclusions: Our results show that the four Gasterophilus species have a high level of genetic diversity and different degrees of genetic differentiation and gene flow among different populations of the same species, reflecting their potential to adapt to the environment and the environmental impact on genetic structure. Knowledge of the genetic structure, population history, and migration will help understand the occurrence and prevalence of gasterophilosis and provide a basis for controlling the local spread of Gasterophilus spp. [ABSTRACT FROM AUTHOR]