ABSTRACTMycoplasma mastitiscan be highly contagious, unresponsive to treatment, and cause severe economic problems in affected herds. Notable routes ofMycoplasmaspp. transmissions are contaminated milking equipment and animal contact through respiratory secretions. Only a few studies report the environment as a possible source of infection. Our group studied the presence of pathogens in houseflies (Musca domestica) in a New York State dairy in the United States. Among others, aMycoplasmaspp. was found in the gut of a housefly captured in the sick pen and identified asM. arginini. Here, we characterized its genome and investigated its relatedness with eight isolates from milk, one isolate from lung tissue collected in the same dairy, and five other dairies in New York State. We applied whole-genome sequencing and phylogenetic analysis based on the sequences of the 16S rRNA gene and 76 conserved proteins. We also assessed anin silicovirulence profile by considering a panel of 94 putative virulence genes. As a result of the genome analysis, the houseflyM. argininiisolate was highly similar to the milk isolates; interestingly, the similarity was highest withM. argininiisolated from milk on the same dairy farm where the housefly was captured. The housefly and milkM. argininiisolates possessed 54 of the 94 pathogenicity genes considered. Our data support the hypothesis that houseflies are carriers ofMycoplasmaspp. and can be considered within the possible roots of environmental transmission of infection in dairy cows. Nevertheless,M. argininipathogenicity will need to be investigated with dedicated studies.IMPORTANCEIt is critical to control the spread of bovine mastitis caused byMycoplasmaspp., as this disease can be highly contagious and have a severe economic impact on affected dairies. A better understanding of possible transmission routes is crucial for infection control and prevention. Based on our data, the composite milk isolates are genetically similar to the housefly isolate. This provides evidence that the sameMycoplasmaspecies found in milk and associated with mastitis can also be isolated from houseflies captured in the dairy environment.