Fusarium head blight (FHB), mainly incited by Fusarium graminearum Schwabe, has caused great losses in grain yield and quality of wheat and barley globally. Fhb7, a gene with a major effect on FHB resistance, was recently cloned and found to encode a glutathione S-transferase (GST). Fhb7 originated from 7E chromosome of Thinopyrum ponticum and confers broad resistance to Fusarium species. However, the high-throughput diagnostic markers are not available for breeding selection, which hampers wide deployment of Fhb7 in breeding programs. To develop such DNA markers for high-throughput screening of the Fhb7, we designed two kompetitive allele specific PCR (KASP) markers (KASP-GST1 and KASP-GST2) based on the promoter and coding sequences of GST homologs and both markers cosegregated with Fhb7 FHB resistance in a mapping population. To validate their usefulness in marker-assisted selection (MAS) in breeding programs, the two codominant gene markers were validated to be diagnostic for Fhb7 in another biparental mapping population and one natural panel. Analysis of 244 Thinopyrum accessions using the two diagnostic gene markers identified only six accessions with the Fhb7 resistance allele, and three of them were cytologically confirmed to be Th. Ponticum, the species in which Fhb7 was originally identified. In addition, sequencing GST homologs in selected samples from five Thinopyrum species identified three haplotypes of GST. The development of the diagnostic markers in this study will facilitate the deployment of Fhb7 in wheat breeding programs to improve FHB resistance.