Efavirenz (EFV) is a widely used antiretroviral, due to its safety, efficacy, and low cost. However, plasma concentrations have been related with an increased risk of virological failure and the appearance of serious adverse reactions. EFV is metabolized by Cytochrome P450, the main isoenzyme involved is CYP2B6 and the most relevant genetic polymorphisms found in several populations has been the CYP2B6 516G> T. The aim of this study was to identify the frequency of the CYP2B6 516G>T polymorphism and its effect on the plasma concentration of efavirenz (EFV) in a group of people living with HIV (PLWH) and undergoing EFV treatment in Morelos, Mexico. Ninety-six PLWH undergoing EFV treatment, at a daily dose of 600 mg orally in combination with other antiretrovirals (ARVs), were included in this study. The CYP2B6 516G>T polymorphism was detected using PCR-RFLP. The plasma concentrations of EFV were evaluated by high-resolution liquid chromatography coupled to a mass-mass detector, using a protein precipitation method. The median plasma EFV concentration was 4.6 μg/mL (IQR = 4.64) and 64.6% of the subjects had concentrations above the therapeutic range. The CYP2B6 516G>T genotype findings were as follows: 46.9% of the population presented the wild-type genotype (GG), while 45.8 % and 7.3 % showed the heterozygote (GT) and the polymorphic homozygote (TT) genotype, respectively. The homozygote G had the lowest plasma concentrations of EFV (median = 4.1 μg/mL and IQR = 1.7 μg/mL), followed by those with the GT genotype (median = 5.1 μg/mL and IQR = 3.0 μg/mL). Participants with the homozygous T genotype had the highest EFV concentrations (median = 9.7 μg/mL and IQR = 5.8 μg/mL). In conclusion, the CYP2B6 516G>T polymorphism was associated with plasma levels of EFV in PLWH undergoing ARV treatment. EFV plasma concentrations at 600mg doses were outside the therapeutic range in most subjects.