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There are limited data on exposure to mycotoxins in Pakistan. Here, we measured exposure to deoxynivalenol (DON), a common contaminant of wheat, and aflatoxin B 1 (AFB 1 ), a known contaminant of rice, using biomarkers of exposure. Wheat ( n = 195) and rice ( n = 62) samples were analyzed for AFB 1 and DON levels, and the corresponding urinary biomarkers were analyzed in urine samples from a rural population ( n = 264, aged 4-80 years, male 58%) using ultra-sensitive liquid chromatography-tandem mass spectrometry. AFB 1 was detected in 66% of rice (5.04 ± 11.94 µg/kg) and 3% of wheat samples. AFM 1 (hydroxylated form of AFB 1 )was detected in 69% of urine samples, mean 0.023 ± 0.048 ng/mL and DON was detected in 20% of urine samples, mean 0.170 ± 0.129 ng/mL. The maximum probable daily intake for DON derived from the urinary biomarker was 59.8 ng/kg b.w./day, which is below the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives' tolerable daily intake (1000 ng/kg b.w./day). However, for aflatoxin, the derived margin of exposure (MoE) of (13.2) was well below the safe MoE (10,000) suggested by the European Food Safety Authority. The calculated aflatoxin-associated cancer risk of 0.514/10 5 individuals/year suggests that measures should be taken to reduce the AFB 1 contamination in food, particularly rice, in Pakistan.