Sclerotinia stem rot (Sclerotinia sclerotiorum) is a major disease of Brassica oilseeds. As suitable donors to develop resistant cultivars are not available in crop Brassicas, we introgressed resistance from a wild Brassicaceae species, B. fruticulosa. We produced 206 B. juncea-B. fruticulosa introgression lines (ILs). These were assessed for pollen grain fertility, genome size variations and resistance responses to Sclerotinia following stem inoculations under disease-conducive conditions. Of these, 115 ILs showing normal fertility and genome size were selected for cytogenetic characterization using florescent genomic in situ hybridization (Fl-GISH). B. fruticulosa segment substitutions were indicated in 28 ILs. These were predominantly terminal and located on B-genome chromosomes. A final set of 93 highly fertile and euploid (2n = 36) ILs were repeat-evaluated for their resistance responses during 2014–15. They were also genotyped with 202 transferable and 60 candidate gene SSRs. Association mapping allowed detection of ten significant marker trait associations (MTAs) after Bonferroni correction. These were: CNU-m157-2, RA2G05, CNU-m353-3, CNU-m442-5, ACMP00454-2, ACMP00454-3, EIN2-3-1, M641-1, Na10D09-1 and Na10D11-1. This is the first time such a molecular mapping technique has been deployed with introgression lines carrying genomic segments from B. fruticulosa, and the first to show that they possess high levels of resistance against S. sclerotiorum.