AbstractCigarette smoking remains an important health concern and is still a leading cause of preventable mortality. Nicotine is the substance responsible for sustained tobacco use and dependence. Identification of biomarkers underlying nicotine dependence behavior is important to identify people at risk for this dependence. In the present study, we identified biochemical and genetic biomarkers of nicotine dependence detected by the Fagerström Test for Nicotine Dependence (FTDN) in Mexican smokers. The nicotine metabolites nicotine‐N′‐oxide, trans‐3′‐hydroxycotinine‐glucuronide (3HC‐O‐Gluc), and nicotine‐N‐Gluc (Gluc) were useful to differentiate nicotine‐dependent from non‐dependent subjects (p < .0001) with an area under the curve (AUC) of 0.7818. Genetic variants in CYP2A6, FMO3, and UGT2B7 (rs2431413, rs28363545, and rs7439326, respectively) were associated with nicotine dependence (p = .03, p = .01, p = .01, respectively). Variations in the enzymatic activity of CYP2A6 were associated with altered nicotine‐N′‐oxide and 3HC‐O‐Gluc levels. Decreased urinary levels of 3HC‐O‐Gluc and increased nicotine‐N′‐oxide were associated with a decrease in the functional activity of CYP2A6. A strong positive correlation was observed between the ratio of urinary 3HC/cotinine, a measure of CYP2A6 activity, and the levels of 3HC‐O‐Gluc (p < .0001, r = .6835), while a strong negative correlation was observed with nicotine‐N′‐oxide (p < .0001, r = .6522) in nicotine‐dependent subjects. No correlations were observed in non‐nicotine‐dependent subjects. These data suggest that particular urinary nicotine metabolites and genetic variants involved in nicotine metabolism are useful to identify subjects with nicotine dependence in the Mexican population.