The ENAM gene is important in the formation of tooth enamel; an alteration can affect the lengthening of the crystals, and the thickness in enamel. The objective was to determine the presence of the single nucleotide variant (SNV) rs12640848 of the ENAM gene in students exposed to different concentrations of fluoride. Methods: A cross-sectional study was conducted on students exposed to high concentrations of fluoride in the city of Durango which were divided according to the severity of fluorosis and dental caries. Genotype determination was performed by DNA sequencing. The relationship between the severity of dental fluorosis and the allele distribution was determined by the Fisher’s exact and Kruskal–Wallis tests. Results: Seventy-one students were included for the sequencing. In the different allelic variations, for the normal genotype AA/TT, the control group presented 75%, for the AG/TC variation, 70.8% in the TF ≤ 4 group, 65% in TF ≥ 5, and 16.7% in TF = 0; with respect to GG/CC variation, 12.5% in TF ≤ 4, 22% in TF ≥ 5, and 8.3% in TF = 0 group (p = 0.000). Conclusion: The ENAM gene showed an association in the population exposed to different concentrations of fluoride.