A total of 13 Aspergillus isolates were collected from different regions of North Eastern parts of Karnataka. Thirteen isolates varied in radial growth from 80-85 mm. Eight isolates produced sclerotia with minimum number (26 cm-2) produced by AF4 isolate and maximum number (119 cm-2) by AF13. They were inoculated on ripened chilli fruits using pin-prick method. Thirteen isolates varied in aflatoxin production from 99.96 to 1639.10 µg/kg as determined by ELISA. AF10 isolate produced maximum potency of 1639.80 µg/kg followed by AF3 with 1195.80 µg/kg and the least production of 99.96 µg/kg was noticed in AF13. All the 13 isolates were amplified for ITS region and were sequenced. Identity of the isolates was confirmed with sequences of NCBI data base of Aspergillus flavus. Further, aflatoxin producing biosynthetic genes were amplified by using two specific primers set AFL and FLA and were confirmed as aflatoxin producing isolates of A. flavus.