Aflatoxin M1 (AFM1) is a secondary metabolite in the breast milk of lactating mothers who consume foodstuffs infected by the fungi Aspergillus flavus and Aspergillus Parasiticus. The concentration of AFM1 in breast milk of lactating mothers is of major public health concern, because it can negatively affect the health of their babies. The major objective of this study was to assess the AFM1 concentration in the breast milk of lactating mothers in Papua New Guinea (PNG). This was a prospective cross-sectional study carried out between 2011 and 2015 in three of the four Regions in PNG: the National Capital District (NCD) in the Southern Region; Eastern Highlands (EHP) and Western Highlands (WHP) provinces in the Highlands Region; and East New Britain (ENB) and Manus provinces in the Islands Region. The Susu Mama, Well-Baby and Paediatric clinics in the General Hospitals in each of the selected provinces in the three regions were the primary sites for this study. A solid phase competitive Enzyme-Linked Immunosorbent Assay (ELISA 96 Microwell plates) was used for the quantification of AFM1 in breast milk from consented lactating mothers. A total of 874 lactating mothers and their babies participated in this study. The mean age of the mothers was 28.0 ±5.5 years. The age range of all the babies was 2 to 6 weeks. 76.1% (665/874) of all breast milk samples analyzed had detectable levels of AFM1. The concentration of AFM1 was above 10.00ppt in 89 (10.2%) of the 874 breast milk samples (which, according to the Australia / New Zealand / Austria safe cut-off limits for AFM1, makes them unsafe for consumption by the babies). The mean AFM1 concentration in the breast milk samples from lactating mothers in EHP (7.99ppt) was higher than that in the samples from the other 4 provinces in the present study. AFM1 concentration was above 10.00ppt in 14 (4.6%) of the 300 breast milk samples from NCD, in 62 (31.0%) of the 200 samples from EHP, in 10 (4.5%) of the 220 samples from ENB and in 3 (3.0%) of the 100 samples from WHP. In order to reduce the AFM1 concentrations in breast milk of lactating mothers, basic nutrition education, aggressive advocacy, social mobilization, awareness campaigns, including communication with all relevant target groups and the relevant policy makers are urgently required.