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The present study was carried out with a specific objective to study postharvest spoilage of Lobhiya (Vigna unguiculata) seeds contaminated with Aspergillus flavus. Infected seeds were collected and cultured on potato dextrose agar (PDA) media, at 25±2 °C. Aspergillus flavus isolates were primarily characterized by its morphological and microscopic characteristics. Collected fungal isolates were also screened for their afaltoxigenic nature on preliminary basis and at molecular level. For preliminary screening, 5 mm disc of fungal culture was soaked with few drops of liquid ammonia. Color change from yellow pigment to plum-red with different intensities showed the mycotoxic nature of the fungus. DNA from fungal isolates was isolated and amplified using PCR with aflatoxin specific primers, apa-2, ver-1 and omt-1. Amplicons of 1032 bp, 895 bp and 596 bp were obtained in most of the isolates regardless of primer set used which was useful to differentiate between mycotoxic and nontoxic isolates of A. flavus. The isolation of aflatoxigenic strains of A. flavus during post-harvest period of lobhiya seeds raise a serious concern over the quality of seeds and a threat to heath of consumers. It was concluded that Aspergillus flavus is responsible for postharvest spolilage of Lobhiya (Vigna unguiculata).