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Search for genic SSRs from transcriptome sequence data of Brassica juncea and B. nigra and from unigene databank of B. rapa resulted in identification of 20,529 SSRs from 330,827 gene sequences. Identified SSRs (named as unigene microsatellite; UGM) were characterized based on their number of repeats, type of motif and base composition. Primers were designed for 2118 UGMs after in silico polymorphism survey and were tested across 11 different genotypes of B. juncea and B. rapa for PCR amplification and polymorphism. It resulted in clear-cut amplification from 1993 UGMs of which 1319 UGMs showed polymorphism. It was observed that a priori in silico polymorphism survey helped in increasing the polymorphism percentage (6.1–13.2) by PCR amplification. We also report here the development of first SSR-based linkage map of Brassica juncea using a DH mapping population derived from F1 of the cross between the lines EH-2 and Pusajaikisan. The map consisted of 860 markers comprising 462 UGMs, 157 BAC-derived SSRs and 241 intron polymorphic markers and covered a total genetic length of 2073.6 cM. The study also reports an interesting observation of appearance of novel non-parental bands in the mapping population from 18 SSRs and 13 IP markers. Search of Arabidopsis genes orthologous to their primer sequences showed that 11 of such Arabidopsis genes code for proteins involved in various abiotic stresses. The highly transferable genic SSRs developed in the study would serve as valuable resources for comparative genome mapping, mapping of qualitative and quantitative traits, gene cloning and marker-assisted breeding in Brassica species.