Sour rot is a complex disease of grapes caused by an interaction of yeast, acetic acid bacteria, and Drosophila spp. Application of insecticides (most commonly zeta-cypermethrin) targeting Drosophila has previously provided substantial control of sour rot in wine grapes of New York vineyards. In harvest season of 2018, a control failure of sour rot and high populations of Drosophila, mostly Drosophila melanogaster, were observed in a vineyard in the Finger Lakes region, NY, despite repeated applications of zeta-cypermethrin (Mustang Maxx).To determine if resistance was responsible for the control failure, we quantified the toxicity of zeta-cypermethrin and the four other insecticides registered for Drosophila control in NY vineyards. Diagnostic concentrations (susceptible strain LC₉₅, 4 × LC₉₅, and 16 × LC₉₅) were used to evaluate percentage survival of the field flies relative to the susceptible Canton-S strain. Resistance to zeta-cypermethrin, acetamiprid, and malathion, but not to spinosad and spinetoram, was observed in the field-collected flies. This study provides evidence that insecticide resistance of Drosophila is associated with control failure of sour rot in some vineyards, and directly influencing grape production. The implications of these results to insecticide resistance monitoring and management are discussed.