PCR based methods are considered as the most accurate and reliable for early diagnosis of food-borne fungal pathogens when compared to the phenotypic detection methods. Aspergillus species included in section Nigri often infects food materials such as grapes, onions cereals, coffee and their derivatives, principally in warm and humid climatic conditions. Here, we propose, a rapid molecular method for detection and discrimination of A. niger using Real-Time PCR SYBR green melt curve analysis. The coding region of AAA ATPases gene fragment was amplified using specific primers and the product was separated based on the melting temperature. The measured melting temperature of A. niger isolates were in the range between 85.16 °C and 85.31°C.