Dissemination of antimicrobial resistance is a major global public health concern. To clarify the role of flies in disseminating antimicrobial resistance between farms, we isolated and characterized tetracycline-resistant Escherichia coli strains isolated from flies and feces of livestock from four locations housing swine (abattoir, three farms) and three cattle farms. The percentages of isolates from flies resistant to tetracycline, dihydrostreptomycin, ampicillin, and chloramphenicol (80.8%, 61.5%, 53.8%, and 50.0%, respectively) and those from animal feces (80.5%, 78.0%, 41.5%, and 46.3%, respectively) in locations housing swine were significantly higher than those from cattle farms (p<0.05). The rates of resistance in E. coli derived from flies reflected those derived from livestock feces at the same locations, suggesting that antimicrobial resistance spreads between livestock and flies on the farms. The results of pulsed-field gel electrophoresis (PFGE) analysis showed that, with a few exceptions, all E. coli isolates differed. Two pairs of tetracycline-resistant strains harbored similar plasmids with the same tetracycline-resistance genes, although the origin (fly or feces), site of isolation, and PFGE patterns of these strains differed. Therefore, flies may disseminate the plasmids between farms. Our results suggest that flies may be involved not only in spreading clones of antimicrobial-resistant bacteria within a farm but also in the widespread dissemination of plasmids with antimicrobial resistance genes between farms.