Soil-borne wheat mosaic virus (SBWMV) is an important viral disease in US, and it can practically destroy the entire crop of a susceptible cultivar. The virus can survive in soil for decades until a suitable host plant is encountered. Utilization of resistant varieties is currently the only practical and environmentally friendly means for controlling SBWMV. However, development of resistant cultivars is slow due to difficulties in germplasm screening in field. Therefore, there is a need to identify tightly linked markers to SBWMV resistance gene(s), so that quick and accurate laboratory-based marker-assisted selection can be used in developing resistant cultivars. Two co-dominant SSR markers Xbarc161 and Xbarc177, closely linked with SBWMV resistance in two separate RILs populations, were previously identified. In this study, 75 advanced lines in 2010 Gulf Atlantic Wheat Nursery (GAWN), and 10 cultivars or lines with Pioneer 26R61 (resistant) or AGS 2000 (susceptible) in their pedigree, were used to validate the marker effectiveness. Pioneer 26R61 and AGS 2000 were positive and negative checks, respectively. The result showed that the genotype were consistent with the phenotype data in the field for 72 lines out of 75. Only 2 lines were negative for the two markers, but showed resistant in the field, therefore, new gene(s) might be present in the two lines. One line was positive for markers, but susceptible, which might be explained by recombination between the markers and the gene. In addition, the marker data was in total agreement with the phenotypic data for 10 cultivars or lines derived from Pioneer 26R61 or AGS 2000. Consequently, Xbarc161 and Xbarc177 were diagnostic for detection of SBWMV resistance, and also could be used as a starting point for discovering new resistant genes for SBWMV in wheat.