Ochratoxin A (OTA) is a naturally occurring fungal metabolite produced by some species of Aspergillus and Penicillium. OTA being nephrotoxic, carcinogenic, hepatotoxic and immunosuppressive imparts many human and animal health hazards. In this study OTA production was observed at different temperatures (5ºC, 10ºC, 20ºC, 30ºC and 40ºC) in three substrates (corn, rice and wheat) by A. ochraceus and A. sulphureus. These two fungal species were grown on aforementioned substrates for three weeks separately. It was quantified by High Performance Liquid Chromatography (HPLC) using the florescent detector. The samples were considered positive for OTA if the peak was obtained at the retention time of 6.34 minutes. OTA was produced at all studied temperatures and in all substrates but the maximum OTA production was observed (1.87±0.021 µg/g) at 30ºC in corn by the Aspergillus ochraceus where as it was minimum at 5ºC in wheat by both species i.e. A. ochraceus (0.19±0.021 µg/g) and A. sulphureus (0.09±0.046 µg/g). Our results indicate that production of OTA by both species of fungus A. ochraceus and A. sulphureus depends upon temperature and substrate and the production in cereals was significantly higher than the maximum permitted level in cereals (5ng/g) as described by the European Union regulation commission and other agencies in the world.