Aflatoxin M1 (AFM1) is the hydroxylated metabolite of aflatoxin B1 (AFB1) formed in the liver of mammals and subsequently excreted in breastmilk (BM) of mammals. Humans are exposed to AFM1 mainly through the consumption of aflatoxin contaminated milk and foods. Determining levels of AFM1 is of significant public health importance due to the risk of exposure to breastfeeding infants. The objective of this study is to determine the prevalence of AFM1 in BM and assess factors associated with BM AFM1 levels in mothers. Data, collected between July 2015 and July 2017, were derived from the Aflacohort study (n = 1675) in Banke district, of Province 5 in the south-west region of Nepal. AFM1 was analyzed using ultra-high-performance liquid chromatography with a lower limit of detection of 0.04 ng/L (ng/L). Multivariate linear and quantile regression models were used to examine the relationship of factors with AFM1, including mother's age, food consumption and body mass index, as well as, seasonality and wealth quintiles. Approximately 94% of BM samples had detectable levels of AFM1 (geometric mean 0.78 ng/L and range 0.04 ng/L to 315.99 ng/L). The consumption of particular foods such as yogurt, milk, hydrogenated oil and ripe pumpkin was positively associated with BM AFM1 levels (p-value <0.05), while consumption of legumes was associated with lower BM AFM1 levels (p-value <0.05). Seasonal influences were significant, with BM collected in the pre-winter and winter seasons exhibiting significantly higher AFM1 levels compared to the spring (p-value <0.05). These results suggest near-ubiquitous presence of aflatoxin in the food supply of this part of Nepal, and possible exposure of AFM1 to infants through BM of mothers. The findings justify integrated strategies across the value chain and at household level to identify dietary sources of aflatoxin exposure, proper post-harvest management and monitoring for aflatoxin control and mitigation.