Background: Non-alcoholic fatty liver disease (NAFLD), the most common chronic liver disease in adolescents, is a feature of metabolic syndrome (MetS). Obesity and insulin resistance (IR) are risk factors for NAFLD, as well as inflammation-related genetic markers. The relationship between metabolic or inflammation-related genetic markers and alanine aminotransferase (ALT) is not fully understood. We examined the relationship of MetS, metabolic and inflammation-related genetic markers with elevated ALT in adolescents. Methods: A total of 674 adolescents participated in a cross-sectional study in Guadalajara, Mexico. Elevated ALT (>40 IU/L), a surrogate marker of NAFLD, and MetS (International Diabetes Federation definition) were evaluated. Obesity, IR, lipids, C-reactive protein (CRP) and genetic markers (TNFA-308G>A, CRP+1444C>T, IL1RN and IL6-597/-572/-174 haplotype) were evaluated. Multivariate logistic regression was performed. Results: Elevated ALT was observed in 3% and 14.1% (total and obese, respectively) of the adolescents. Obesity (odds ratio [OR], 5.86; 95% confidence interval [95% CI], 1.16–25.89), insulin (OR, 8.51; 95% CI, 2.61–27.71), IR (OR, 9.10; 95% CI, 2.82–29.38), total cholesterol (TC) (OR, 3.67; 95% CI, 1.25–10.72), low-density lipoprotein-cholesterol (LDL-C) (OR, 3.06; 95% CI, 1.06–8.33), non-high-density lipoprotein-cholesterol (HDL-C) (OR, 3.88; 95% CI, 1.27–11.90) and IL1RN (OR, 4.64; 95% CI, 1.10–19.53) were associated with elevated ALT. Among males, ≥2 MetS criteria were associated with elevated ALT (OR, 4.22; 95% CI, 1.14–15.71). Conclusions: Obesity, insulin, IR, high TC, high LDL-C, high non-HDL-C and IL1RN polymorphism were associated with elevated ALT. Among males, ≥2 MetS criteria were associated with elevated ALT. There is an urgent need to reduce obesity and IR in adolescents to prevent NAFLD.