Objective To investigate the current insecticide resistance situation of Musca domestica in different habitats in Wuhan, to provide scientific basis for rational application of insecticides and effective control. Methods The micro-drop method was applied to test medium lethal dose (LD₅₀) of 3-4 day old female M. domestica to pesticides. Kruskal-Wallis H Rank Sum Test and Mann-Whitney U Rank Sum Test were used for statistical significance analysis. Results In residential areas, farmers' markets and refuse transfer stations, M. domestica showed high resistance to cypermethrin, deltamethrin and methylpyridine. The LD₅₀ of the M. domestica ranged from 0.095 to 0.685 μg per fly, and resistance ratios were 26.36 to 710.00-fold, there was no significant difference (χ²=0.007, 0.284, 1.280, 0.152, P=0.996, 0.868, 0.527, 0.927). Musca domestica in residential areas and farmers' markets showed low resistance to amethrin, while M. domestica in refuse transfer stations were susceptible to cypermethrin, with statistical significance (χ²=6.292, P=0.047). The results showed that the resistance level of M. domestica to cypermethrin in refuse transfer station was significantly lower than that in residential area (U=8.000, P=0.033) and farmers market (U=7.500, P=0.028), but there was no significant difference between residential area and farmer market (U=24.000, P=0.949). Conclusion The M. domestica strains in three different habitats in Wuhan demonstrated high resistance to beta-cypermethrin, cypermethrin, deltamethrin and azamethiphos, to which the difference among the susceptibility of the M. domestica strains from three different habitats was detected. Therefore, it is suggested to take integrated control measures and apply insecticides scientifically and rationally to avoid the development of pesticide resistance in M. domestica.