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Abstract Background & aim: Aflatoxins are a large group of mycotoxins. The aim of the present study was the comparison of Aflatoxin B1 (AFB1) production by Aspergillus flavus (IR 111) and Aspergillus parasiticus (NRRL 2999) under various conditions of temperature, light, and pH. Methods: In this experimental study, twenty-four flasks were assigned for incubation of each of the fungi A. Flavus and parasiticus at 18, 24, 32 °C. Both flasks were maintained under conditions of light and darkness. The rate of (AFB1) produced by each groups, was measured by thin layer chromatography. Data were analyzed using descriptive statistical analysis (SPSS, version 16). Results: The lowest yield of (AFB1) produced by A. Flavus and parasiticus belonged to 32 °C and pH 6.5, respectively. On the other hand, the highest yield of toxin was observed at 24 °C and pH 6. The lighting effects were considerable. According to the studies on the adverse effects of light and the fermentation process, aflatoxin production increased in dark conditions. Conclusion: The results of study showed that A. Parasiticus (NRRL 2999) produced more aflatoxin than A. Flavus (IR 111). Key words: Aflatoxin B1, Aspergillus flavus, Aspergillus parasiticus