A total of 119 samples of peanut were collected throughout the peanut production chain in São Paulo State, Brazil. The peanut samples were directly plated for determination of percentages of infection and a polyphasic approach was used to identify Aspergillus section Flavi species. Further, the potential for aflatoxin production by the isolates was tested using the agar plug technique and the presence of aflatoxins in peanuts was assessed using an immunoaffinity column followed by quantification using HPLC with reverse phase column and fluorescence detection. The limit of detection and quantification were 0.05 and 0.17μg/kg for total aflatoxins, respectively. Four species of Aspergillus section Flavi were isolated: A. caelatus (11), A. flavus (515), A. parasiticus (17) and A. tamarii (13). All isolates of A. parasiticus were able to produce aflatoxin B and G whereas aflatoxin B was produced by 50% of A. flavus isolates. Aflatoxins were found in 12 samples at concentrations ranging from 0.3 to 100μg/kg. The data reported in this study add information on the occurrence and biodiversity of fungi in peanuts at several stages of the production chain. The occurrence of aflatoxins is also of major relevance for continuous monitoring and assessment of likely exposure of consumers to aflatoxins through consumption of peanuts.