Abstract Importance: Southern Mexico and Guatemala, together represent the region with the highest liver cancer burden in the Americas. There is a higher burden of liver cancer in rural regions in eastern and southern Mexico and remarkably there is an equal number of cases in men and women. Reasons for this increased burden are not clear, but previous studies of food samples have found that contamination of maize with aflatoxin B1 (AFB1), a potent liver carcinogen, is common in the region while HBV and HCV seroprevalence is less than 0.5%. Few examinations of AFB1 levels in persons have been conducted. Objective: To estimate the prevalence of exposure and circulating levels AFB1 in a representative sample of adults living in five regions across Mexico. Study Design: We randomly selected a subsample of 955 adults ≥40 years who participated in Mexico’s National Health and Nutrition Survey 2018 (ENSANUT). These residents were from five states in eastern and southern Mexico and all provided a venous blood sample. We assessed AFB1 levels as pg AFB1-lys/µL in serum using mass spectrometry. The limits of detection and quantification were both 0.010 pg/μL. Results: The mean age of participants was 56 years (± SD 11.7) and 54% were female. The overall prevalence of AFB1-lys detection was 92% (95% confidence intervals; 95%CI 88, 94%). The region with the lowest prevalence, 88% (95%CI 78, 94%), was Chiapas and the region with the highest prevalence was Veracruz 95% (95%CI 84, 98). Median AFB1 was 0.127 pg AFB1-lys/µL (interquartile range; IQR 0.054, 0.446) with a range from 0.066 (IQR, 0.033, 0.147) in Yucatán to 0.476 (IQR, 0.199, 1.355) in Veracruz. AFB1 exposure levels were higher in persons aged ≥60 years, men, and participants living in rural areas. We observed decreasing levels of AFB1 exposure with increasing socioeconomic status. Conclusions: In a representative sample of adults in eastern and southern Mexico we observed a high prevalence of exposure to AFB1. For certain regions, AFB1 levels are comparable to those in Guatemala. These results suggest that AFB1 may be related to the increased burden of liver cancer in eastern and southern Mexico. Citation Format: Adriana Monge, Katherine McGlynn, Luis Santiago-Ruiz, Jose Maria Remes-Troche, Karina Hernandez-Flores, Salvador Villapando-Hernandez, Martin Romero, John Groopman, Martin Lajous. Aflatoxin exposure in a population-based representative sample of adults in Mexico in 2018. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4221.