Yellowing disease, caused by viral infection, is a problem in sugar beet production. In Japan, yellowing disease caused by infection with Beet leaf yellow virus (BLYV), similar to Beet western yellows virus, occur periodically, and result in low yields. Therefore, development of varieties resistant to yellowing disease is desired. Previously, we found QTLs linked to yellowing disease resistance using a genetic resource of USDA. In this study, we tested the effectiveness of molecular markers linked to the QTLs for selection of yellowing resistant breeding line. We crossed our O-type (maintainer of Owen CMS) lines susceptible to yellowing disease, with resistant genetic resources of USDA to create segregating populations. Their resistance to yellowing were tested by inoculation test of BLYV via peach aphids. The yellowing index (YI) of susceptible O-type were above “2.5” (0: no disease symptom to 3: 100% yellowing of mature leaves), whereas the resistant genetic resource had YI less than “1.0”. The F3 lines showed variation in YI ranging from “0.3” to “2.9”. Genotyping of markers linked to resistance revealed that the F3 lines having resistant genotype of the markers averaged YI of “0.7”, whereas F3 lines having susceptible genotype of the markers averaged YI of “2.0”. These results demonstrated that the markers can effectively select yellowing resistance. These lines were further selected for sugar content, restorer-of-fertility genes, and monogerminity to obtain O-type candidates. The agronomic characteristics of these lines will be determined in the future. This research was supported by the research program on development of innovative technology grants from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN).