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There are no studies that have utilized both biomarkers and self-reported data to evaluate maternal alcohol use during pregnancy in Mexico. Therefore, we aimed to describe the prevalence of alcohol consumption in a cohort of 300 Mexican pregnant women. We used a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to measure hair ethyl glucuronide (EtG) in hair segments that corresponded to the first and second half of pregnancy. We compared the hair EtG values to a self-reported questionnaire on maternal drinking habits and evaluated whether the gestational alcohol use was associated with psychotropic drug use. Based on the EtG measurements, 263 women (87.7%) were alcohol-abstinent during the entire pregnancy, while 37 (12.3%) had used alcohol at least once during the pregnancy. Of these, only two women were found to have problematic alcoholic behavior during the entire pregnancy. No significant differences in sociodemographic characteristics were observed between alcohol-abstinent women and women with drinking habits. The self-reporting data and hair EtG gave heterogeneous results: although 37 women had self-reported alcohol use during pregnancy, only 54.1% of these women tested positive for hair EtG. Of the women who tested positive for hair EtG, 54.1% tested positive for psychoactive substances. In our cohort, the use of drugs of abuse was independent of gestational drinking. This study provided the first objective evidence of prenatal ethanol consumption in a cohort of Mexican pregnant women., Competing Interests: Declaration of competing interest None to disclose., (Copyright © 2023 Elsevier Inc. All rights reserved.)