The productivity of Indian mustard Brassica juncea L., a major oilseed crop in India, is heavily inflicted due to the fungal disease Alternarialeaf spot caused by Alternaria brassicae L. Genetic resistance against this disease is not available in the mustard cultivars. Screeningfor resistance among the cruciferous wild relatives identified Diplotaxis erucoides L. as a resistant species that can be used for sourcingresistance genes against Alternaria disease. The introgression lines developed by wide hybridization between D. erucoides and B. junceahave been advanced to BC2F7 generation through a recurrent selection of resistant plants at three locations. Development of molecularmarkers linked to alien introgression and the trait is highly desirable for tracking the alien introgression and early selection of thedesired plants. A study was conducted to develop STS markers associated with the introgressed alien DNA based on resequencing of aresistant and a susceptible introgression line. A step-wise bioinformatic analysis and identification of the species-specific DNA contigsfollowed by annotation led us to predict several putative STS markers. While the sequence data generated from more resistant plantsand further analysis are in the process, initial wet lab validation endorsed the appropriateness of the strategy. The identified markersthat showed species specificity need to be studied over the generations to establish their linkage with the trait of Alternaria resistance.