Singh, B. K., Nandan, D., Supriya, A., Ram, B., Kumar, A., Singh, T., Meena, H. S., Kumar, V., Singh, V. V., Rai, P. K. and Singh, D. 2015. Validation of molecular markers for marker-assisted pyramiding of white rust resistance loci in Indian Mustard (Brassica juncea L.). Can. J. Plant Sci. 95: 939-945. Successful application of molecular markers in marker-assisted pyramiding relies on effective determination of the target phenotype. In this respect, evaluation of the efficiency of markers for marker-assisted selection through cross-validation in different genetic backgrounds and in different populations is a crucial step. In the present study, the previously identified Arabidopsis-derived intron polymorphic (IP) markers At5g41560 and At2g36360, which were highly linked with AcB1-A4.1 and AcB1-A5.1, respectively, were validated in a set of 25 genotypes of Indian Mustard and in three different F₂ populations. The relationships between the variation of PCR products of the two markers with the percent disease index (PDI) of the tested genotypes, and the co-segregation analysis of the markers with disease phenotype in F₂ populations clearly indicated that At5g41560 and At2g36360 are genotype-nonspecific markers and are closely linked to white rust resistance loci AcB1-A4.1 and AcB1-A5.1, respectively. It also became evident from the present study that AcB1-A4.1 and an another white rust resistance locus Ac(2)t are likely the same gene locus.