Brassica juncea has adapted to diverse climate zones and latitudes, especially in the Yunnan–Guizhou Plateau with the complexity and diversity of the ecological types of western China. However, the genetic variations underlying the diversity of these ecotypes are poorly known. In this study, we resequenced the genome of 193 indigenous B. juncea accessions and obtained 1.04 million high-quality SNPs and 3.23 million InDels by mapping reads to the reference genomes of B. juncea var. timuda. Phenotype, population genetic, phylogenetic and principal component analyses showed considerable genetic variation including four distinct genetic groups. Selective sweep analysis and a genome-wide association study revealed the candidate genes for seed color and fatty acid biosynthesis. The results provide a comprehensive insight into the spread and improvement of B. juncea and laya foundation for accelerating rapeseed breeding by facilitating screenings of molecular markers.