All species posses detoxification pathways enabling them to respond to environmental toxins. Given the widespread use of pesticides, and the frequent development of resistance to these compounds by crop pests, it is critical to understand the genetic basis of xenobiotic resistance in insects. Previously, we used the Drosophila Synthetic Population Resource to map four QTL contributing to resistance to nicotine, a compound employed as a natural insecticide by some plants. RNA-seq showed that both cytochrome P450 genes under QTL1, Cyp28d1 and Cyp28d2, and one of ten UDP-glucuronosyltransferase (Ugt) genes under QTL4, Ugt86Dd, were differentially-expressed between susceptible and resistant strains. Here we carry out RNAi, quantitative complementation tests, overexpression experiments, and mutant analysis to functionally validate the effects of these loci on nicotine resistance, and attempt to identify causative sequence variants. Ubiquitous RNAi knockdown of Cyp28d1 and Ugt86Dd, as well as targeted knockdown in the anterior midgut, reduced nicotine resistance, providing evidence these genes are involved in the phenotype. To determine whether these loci harbor segregating variation influencing resistance we crossed susceptible and resistant strains to a range of deficiencies and insertional mutants. These quantitative complementation tests revealed functional allelic variation at Cyp28d1 and Cyp28d2, and suggested that multiple nicotine resistance factors are present within the QTL4 region, consistent with the idea that several of the Ugt genes are involved. Sequencing the Ugt86Dd open reading frame revealed a 22bp coding deletion segregating in our multi-parental mapping panel, with the four most susceptible founder haplotypes at the QTL all harboring the deletion allele. We constructed overexpression genotypes using both Ugt86Dd alleles, and found that nicotine resistance is significantly greater following anterior midgut overexpression of the insertion allele compared to the deletion allele, implying the variant has a functional role. To further test the effect of this polymorphism we generated two mixed populations derived from Drosophila Genetic Reference Panel; one founded with all seven DGRP strains homozygous for the deletion allele, and one founded with a random seven strains homozygous for the insertion. Flies from the population fixed for the deletion allele were more susceptible for nicotine, supporting the idea that this variant directly contributes to nicotine resistance. We have now successfully generated custom Ugt86Dd mutants using the CRISPR-Cas9 system, and future experiments will help elucidate the effect of the locus on nicotine resistance.