The organotin acaricide fenbutatin oxide (FBO) - an inhibitor of mitochondrial ATP-synthase - has been one of the most extensively used acaricides for the control of spider mites, and is still in use today. Resistance against FBO has evolved in many regions around the world but only few studies have investigated the molecular and genetic mechanisms of resistance to organotin acaricides. Here, we found that FBO resistance is polygenic in two genetically distant, highly resistant strains of the spider mite Tetranychus urticae, MAR-AB and MR-VL. To identify the loci underlying FBO resistance, two independent bulked segregant analysis (BSA) based QTL mapping experiments, BSA MAR-AB and BSA MR-VL, were performed. Two QTLs on chromosome 1 were associated with FBO resistance in each mapping experiment. At the second QTL of BSA MAR-AB, several cytochrome P450 monooxygenase (CYP) genes were located, including CYP392E4, CYP392E6 and CYP392E11, the latter being overexpressed in MAR-AB. Synergism tests further implied a role for CYPs in FBO resistance. Subunit c of mitochondrial ATP-synthase was located near the first QTL of both mapping experiments and harbored a unique V89A mutation enriched in the resistant parents and selected BSA populations. Marker-assisted introgression into a susceptible strain demonstrated a moderate but significant effect of the V89A mutation on toxicity of organotin acaricides. The impact of the mutation on organotin inhibition of ATP synthase was also functionally confirmed by ATPase assays on mitochondrial preparations. To conclude, our findings suggest that FBO resistance in the spider mite T. urticae is a complex interplay between CYP-mediated detoxification and target-site resistance.