Gray leaf spot (GLS) is a foliar disease of maize caused by Cercospora zeae-maydis and Cercospora zeina and quantitative resistance to GLS is important for maize production. A nested association mapping (NAM) maize population, consisting of 25 populations of 150 recombinant inbred lines, was used to identify quantitative trait loci (QTL) for GLS resistance. Trials were conducted in Blacksburg, VA, in a field with high natural incidence of GLS. A multivariate mixed model was used in ASReml3 to give the best linear unbiased predictions of disease severity ratings. QTL were selected using a general linear model selection procedure in SAS 9.2. Sixteen QTL, distributed across the maize genome, were identified using a likelihood of odds (LOD) selection threshold >4. Seven of these 16 QTL displayed allelic series with significantly higher and lower effects than the common parent allele. Near-isogenic lines (NILs) extracted from heterogeneous inbred families were developed to confirm and further finemap select QTL, targeting the loci with the greatest LOD scores from the model selection QTL analysis. Phenotypic characterization of the NILs confirmed that the loci in bins 1.04, 2.09 and 4.05 likely contribute significantly to disease resistance, with bins 1.04 and 2.09 conferring reductions in disease of 12% and 23%, respectively. In contrast, the susceptible allele in bin 4.05, which was associated with the distance between major veins, conferred an increase of 8.4%. This disease-related venation trait was confirmed using the 4.05 NILs. Genome-wide association studies revealed candidate genes related to the production of carotenoids, anthocyanins and antioxidant compounds that may play a role in cercosporin detoxification. Expression analysis of 1.05 NILs treated with cercosporin implicated a flavin-monooxygenase gene in cercosporin detoxification. Furthermore, significant associations between NAM parental allelic effects and parental phenotypes at the microscopic level for the 1.02 and 1.06 loci implicated callose plug and phenolic accumulation, respectively, in host defense. Elucidating the genetics of quantitative disease resistance loci provides breeders with valuable information that may enhance their ability to use molecular markers as a means to rapidly introgress loci that provide quantitative disease resistance.