Rapeseed mustard (Brassica juncea L.) is the third most important oilseed crop in the world, but the genetic mechanism underlying its massive phenotypic variation remains largely unexplored. In this study, specific length amplified fragment sequencing (SLAF-Seq) was used to resequence a population comprising 197 F8 recombinant inbred lines (RILs) derived from a cross between vegetable-type Qichi881 and oilseed-type YufengZC of B. juncea. In total, 438,895 high-quality SLAFs were discovered, 47,644 of which were polymorphic, and 3,887 of the polymorphic markers met the requirements for genetic map construction. The final map included 3,887 markers on 18 linkage groups and was 1,830.23 centiMorgan (cM) in length, with an average distance of 0.47 cM between adjacent markers. Using the newly constructed high-density genetic map, a total of 53 QTLs for erucic acid (EA), oleic acid (OA), and linolenic acid (LNA) were detected and integrated into eight consensus QTLs with two for each of these traits. For each of these three traits, two candidate genes were cloned and sequence analysis indicated colocalization with their respective consensus QTLs. The co-dominant allele-specific markers for Bju.FAD3.A03 and Bju.FAD3.B07 were developed and showed co-localization with their consensus QTLs and co-segregation with LNA content, further supporting the results of QTL mapping and bioinformatic analysis. The expression levels of the cloned homologous genes were also determined, and the genes were tightly correlated with the EA, OA and LNA contents of different lines. The results of this study will facilitate the improvement of fatty acid traits and molecular breeding of B. juncea. Further uses of the high-density genetic map created in this study are also discussed.