Abstract The house fly, Musca domestica L. (Diptera: Muscidae), is a cosmopolitan pest that poses health risks to humans and domesticated animals. Pyrethroid insecticides target the insect voltage-sensitive sodium channel (VSSC) and have been widely used to control house flies in poultry farms in Argentina for more than 3 decades. The continued use of pyrethroids has resulted in the development of resistance. There are 3 main VSSC mutations known to cause resistance to pyrethroids in house flies: kdr (L1014F), kdr-his (L1014H), and super-kdr (M918T + L1014F). The present study aimed to assess the presence and frequency of VSSC alleles in populations previously identified as exhibiting high resistance to permethrin in Argentina. Adult house flies were collected from 3 poultry farms located in Buenos Aires Province. The total distributions of the susceptible, kdr, kdr-his, and super-kdr alleles were 28.9%, 22.2%, 21.1%, and 27.8%, respectively. Nine of the ten possible genotypes were identified within the studied populations. At least one resistant allele was carried by 87% of the individuals, whereas 71% were heterozygous for 2 resistant alleles. The levels of allelic richness and heterozygosity were high for all populations, and a departure from Hardy–Weinberg expectations was observed in one of them. Our findings are the first to report the presence of kdr, kdr-his, and super-kdr mutations in house flies collected from poultry farms in Argentina. This result indicates that the populations are under active selective pressure exerted by pyrethroids, and new alternatives should be used in integrated control programs.