Vinegar flies are vectors of pathogens causing fruit rots of grapes, so control of these insects is important for preventing vineyard yield loss. Recent outbreaks of sour rots may be linked to greater challenges controlling vinegar flies, so we investigated the insecticide susceptibility of populations collected from commercial vineyards across Michigan. We first determined the discriminating concentration for phosmet, malathion, methomyl, and zeta-cypermethrin using a laboratory susceptible (Canton-S) strain of D. melanogaster females. The discriminating concentrations were determined as 252.08, 2.58, 0.96, and 1.68 ppm of the four insecticides, respectively. These concentrations were first tested in 2020 against populations from the two major counties for grape production. In 2021, we expanded monitoring to twenty-three populations collected from vineyards across six counties. All populations had significantly lower sensitivity to all four insecticides compared with Canton-S strain, with up to 98.8% lower mortality for phosmet. The LC₅₀, LC₉₀, and LC₉₉ values of the four insecticides for the two populations tested in 2020 were 7–1,157-fold higher than the Canton-S strain. For the twenty-three populations collected in 2021, mortality ranged from 56.3 to 100% when the flies were screened using a 10x concentration of the discriminating concentration of the insecticides, whereas it ranged from 82.4 to 100% when the flies were screened using a 20x concentration. Our results suggest variable levels of resistance to insecticides from multiple chemical classes in D. melanogaster populations in Michigan vineyards, highlighting the need to implement integrated sour rot management approaches that are less dependent on insecticides for control of this species.