Groundnut (Arachis hypogaea L.) is one of the most susceptible host crops to Aspergillus flavus (Link Ex Fries)invasion and subsequent aflatoxin production before and after harvest. The present experiment comprised 9 local cultivarsof Karnataka, 34 germplasm collections and 25 advance breeding lines from ICRISAT along with popular tolerant varietyJ-11 used as check. The study was conducted to screen for A. flavus invasion by following the standard method used atICRISAT using the aggressive strain Af (11-4). Ample amount of variation among cultivars and germplasm lines for seedcolonization severity was observed in spore spray as well as in pin prick method of screening. From the present investigation,it is clear that ICGV-02207 and ICGV-02266 exhibited both seed coat and cotyledon resistance towards A. flavus infectionand less aflatoxin accumulation as compared to susceptible one. These can serve as good source of resistance for futuregroundnut breeding.Key words: Aspergillus flavus, Groundnut, Screening, Variety