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Resistance to wheat spike blast (WSB), caused by the Magnaporthe oryzae triticum pathotype (MoT), has relied upon a single major source: the 2NvS translocation introgressed from the wild relative Aegilops ventricosa Tausch. However, this resistance is partial and recently partially overcome by newer MoT races. To characterize potential novel loci conferring resistance to WSB, we conducted a genome‐wide association study (GWAS) using a diverse panel of 384 wheat genotypes phenotyped under three controlled‐environment conditions using MoT isolates T‐25 (301 genotypes), B‐71 (87 genotypes), and 008 (49 genotypes). Genotyping‐by‐sequencing identified 13,175 single nucleotide polymorphisms (SNPs) after filtering. Principal components analysis (PCA) identified two clusters based on the presence or absence of the 2NvS translocation, and the first three PCAs explained 13% of the genetic variation. Three individual analyses were performed (full [all genotypes combined], 2NvS genotypes only, and non‐2NvS genotypes only) using a linear mixed model and a threshold of significance of false discovery rate at 5%. Association analysis detected 25 significant SNPs for the full GWAS with isolate T‐25, in which 21 were mapped on chromosome 2A in the same physical position as the 2NvS translocation. Highly significant linkage disequilibrium among these SNPs suggested they might tag the same quantitative trait locus (QTL). No significant associations were identified with isolates B‐71 and 008, likely due to the small sample size. A QTL pyramiding analysis showed that the combination of multiple QTL was not statistically different from the individual effect of the 2A QTL. Further validation of these genomic regions can aid breeding for broad spectrum and durable WSB resistance. [ABSTRACT FROM AUTHOR]