The widespread disease vector Aedes aegypti presents a severe threat to human populations as a carrier of Zika, dengue, yellow fever, and chikungunya viruses. The primary vector control method involves insecticide use; unfortunately, heavy reliance on insecticides has resulted in the proliferation of insecticide resistance. In addition to mutations in the voltage gated sodium channel that reduce insecticide efficacy, research suggests that metabolic detoxification is a critical resistance mechanism in A. aegypti. Using high-throughput sequencing (HTS), previous research (Saavedra-Rodriguez et al., 2019) identified genome-wide polymorphic sites that were significantly associated with deltamethrin resistance in one A. aegypti population from southern Mexico named Viva Caucel. In the present study, we used Sanger sequencing to validate the existence of single nucleotide polymorphisms (SNPs) in six detoxification genes identified in the HTS study. These SNPs were located in genes encoding for an oxidation-reduction enzyme (Aldox 10391), an esterase (CCEae2B) and four cytochrome p450 oxidases (CYP4H33, CYP325M2, CYP325G3, CYP325L1). We genotyped knockdown-resistant and susceptible Viva Caucel individuals using allele-specific PCR melting curves to quantif each SNP s association with resistance. The HTS and allele-specific PCR methods reported similar allelic frequencies of resistant versus susceptible phenotypes, confirming the value of HTS for estimating allelic frequencies at polymorphic sites and for detecting mutations putatively associated with insecticide resistance. We subsequently examined the same SNPs in another population from southern Mexico named 5 de Febrero. The SNP located in CYP325L1 was significantly associated with resistance in both populations, suggesting that it likely plays a role in the metabolic resistance mechanism. Evaluating detoxification SNPs is critical to identifying potential genetic markers of insecticide resistance and informing future mosquito control strategies.