Gray leaf spot (GLS), caused by Magnaporthe grisea, is a major fungal disease of St. Augustinegrass (Stenotaphrum secundatum), causing widespread blighting of the foliage under warm, humid conditions. To identify quantitative trait loci (QTL) controlling GLS resistance, an F1 mapping population consisting of 153 hybrids was developed from crosses between cultivar Raleigh (susceptible parent) and plant introduction PI 410353 (resistant parent). Single-nucleotide polymorphism (SNP) markers generated from genotyping-by-sequencing constituted nine linkage groups for each parental linkage map. The Raleigh map consisted of 2,257 SNP markers and spanned 916.63 centimorgans (cM), while the PI 410353 map comprised 511 SNP markers and covered 804.27 cM. GLS resistance was evaluated under controlled environmental conditions with measurements of final disease incidence and lesion length. Additionally, two derived traits, area under the disease progress curve and area under the lesion expansion curve, were calculated for QTL analysis. Twenty QTL were identified as being associated with these GLS resistance traits, which explained 7.6 to 37.2% of the total phenotypic variation. Three potential GLS QTL "hotspots" were identified on two linkage groups: P2 (106.26 to 110.36 cM and 113.15 to 116.67 cM) and P5 (17.74 to 19.28 cM). The two major effect QTL glsp2.3 and glsp5.2 together reduced 20.2% of disease incidence in this study. Sequence analysis showed that two candidate genes encoding b-1,3-glucanases were found in the intervals of two QTL, which might function in GLS resistance response. These QTL and linked markers can be potentially used to assist the transfer of GLS resistance genes to elite St. Augustinegrass breeding lines.