Technical Abstract: Aflatoxin, a carcinogenic mycotoxin, remains a major problem throughout the southern USA and World. In this study, diverse, novel maize germplasm from the Genetic Enhancement of Maize (GEM) project was screened to determine the combining ability and inheritance of ear rot resistance and resistance to mycotoxin accumulation in hybrid combinations. Nine GEM germplasm lines and six known resistant and susceptible lines were selected as parents of a diallel cross that was evaluated over two years at Mississippi State, MS, and one year at College Station, TX. Resistant lines, Mp717 and Mp494, each had negative and significant GCA effects, and were frequently present as parents in the hybrid combinations with the lowest aflatoxin accumulation. Mp313E x GEMS-0074 had especially low aflatoxin accumulation across all environments suggesting a useful combination for line development. Of the GEM lines evaluated, GEMN-0130 was the best source of aflatoxin resistance with significant and negative GCA values at all three site years. GEM lines exhibiting significant and negative GCA effects for aflatoxin accumulation in at least one site year include GEMS-0030 (two years) CUBA117 (one year) and GEMS-0074 (one year). GEM lines significantly contributing to reduced ear rot in at least two out of three environments included CUBA117, GEMS-0030, GEMS-0002, and GEMN-0130. These results demonstrate the GEM Project is a useful and untapped source of diverse germplasm for resistance to A. flavus ear rot and aflatoxin accumulation.