AbstractBACKGROUNDFungal infestation is a leading cause of qualitative and quantitative deterioration of stored wheat grains. Limited information is available on the spatial distribution of fungal biota associated with stored wheat grains in India. Fungi were isolated and characterized from nine stored wheat grain samples in three warehouses of the Food Corporation of India, located in three agro‐climatic zones (Paschim Medinipur, Bankura and Purulia) of West Bengal in India.RESULTSMaximum density and fungal diversity were observed in dichloran glycerol agar (DG‐18) medium and the number increased with the increase of storage duration. Samples collected from Purulia showed maximum fungal diversity than that from Bankura and Paschim Medinipur. A total of 284 fungal isolates were obtained, classified into 29 operational taxonomic units (based on amplified ribosomal DNA restriction analysis of 18S and internal transcribed spacer sequences), and identified as 24 different fungal species. The majority of fungal isolates belonged to Aspergillus flavus (35%) followed by Rhizopus oryzae (13%) and Eurotium amstelodami (9%). Aspergillopepsin O (PEPO) gene and aflatoxin biosynthetic pathway gene, nor‐1, were amplified by polymerase chain reaction (PCR) from 91% and 71% of Aspergillus flavus isolates, respectively, indicating their aflatoxin producing ability. Aflatoxin production was further confirmed by ammonia vapour test, thin layer chromatography (TLC) and high‐performance liquid chromatography (HPLC).CONCLUSIONThe presence of toxigenic fungi in stored wheat grain emphasizes the necessity of quarantine measures of stored grains before placing them in the public domain to save consumers from health hazards. © 2019 Society of Chemical Industry