Objective Populations of Mexican American ancestry are at an increased risk for nonalcoholic fatty liver disease. The objective of this study was to determine whether loci in known and novel genes were associated with variation in aspartate aminotransferase (AST) (n = 3,644), alanine aminotransferase (ALT) (n = 3,595), and gamma‐glutamyl transferase (GGT) (n = 1,577) levels by conducting the first genome‐wide association study (GWAS) of liver enzymes, which commonly measure liver function, in individuals of Mexican American ancestry. Methods Levels of AST, ALT, and GGT were determined by enzymatic colorimetric assays. A multi‐cohort GWAS of individuals of Mexican American ancestry was performed. Single‐nucleotide polymorphisms (SNP) were tested for association with liver outcomes by multivariable linear regression using an additive genetic model. Association analyses were conducted separately in each cohort, followed by a nonparametric meta‐analysis. Results In the PNPLA3 gene, rs4823173 (P = 3.44 × 10−10), rs2896019 (P = 7.29 × 10−9), and rs2281135 (P = 8.73 × 10−9) were significantly associated with AST levels. Although not genome‐wide significant, these same SNPs were the top hits for ALT (P = 7.12 × 10−8, P = 1.98 × 10−7, and P = 1.81 × 10−7, respectively). The strong correlation (r2 = 1.0) for these SNPs indicated a single hit in the PNPLA3 gene. No genome‐wide significant associations were found for GGT. Conclusions PNPLA3, a locus previously identified with ALT, AST, and nonalcoholic fatty liver disease in European and Japanese GWAS, is also associated with liver enzymes in populations of Mexican American ancestry.