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The present study was carried out on 110 examined fishes, out of which 70 were diseased fish and 40 healthy. The samples were collected alive from River Narmada at different sites in Indore from March to November 2019. Grossly examined diseased fish revealed different sized ulcers, eroded fins, redness on the skin, eye turbidity, protruded anal and de-scaling of skin, especially in fish naturally infected with Aspergillus, Saprolegnia, mucor, Penicillium and Alternaria. Mycological examination revealed isolation of 9 genera of fungi from a total incidence of 260 fungal isolates, 210 fungal isolates from diseased fish and 50 isolates from an apparently healthy one. Aspergillus flavus and Aspergillus niger were the most predominant fungi isolated from either apparently healthy or even diseased internal organs and from external wounds of fish with Sabouraud Dextrose Agar (SDA) medium. Molecular identification was performed using the inter-transcribed spacer (ITS) gene for Aspergillus. Developed PCR assay specific primer detect A. flavus and A. niger showing clear bands at 595 and 600 bp molecular weight, respectively.