In the present study, analyses of SSR molecular markers were performed to investigate the genetic diversity of 133 tuber mustard cultivars. Eighty-one pairs of SSR primers from a total of 600 in Brassica produced stable amplified bands. In addition, 810 bands were detected among the cultivars, and 724 of those were polymorphic (89.38 %). The average number of bands per locus was 10.0 with a range from 5 to 16. Shannon’s information index for each SSR locus varied from 0.52 to 3.72, with an average of 2.74. The coefficients of genetic similarity in the SSR marker patterns among the 133 cultivars ranged from 0.77 to 0.91, with an average of 0.85. The cluster analysis showed that the cultivars could be classified into six clusters when the genetic similarity was 0.83, with 90.23 % of the cultivars included in Clusters 5 and 6. Principal component analysis was carried based on the SSR data. The results showed that the first three principal components could explain the genetic variation with 85.47, 0.67, and 0.61 %, and the 133 cultivars could be divided into six clusters according to the nearest phylogenetic relationship. It was indicated that the similarity was high and the genetic diversity was narrow among the 133 mustard tuber cultivars. 360 individuals from 24 cultivars were analyzed to reveal the genetic structure and genetic diversity within cultivars. A total of 925 alleles were detected in the 24 cultivars. Estimates of the mean number of alleles ‘A’, the effective allelic number ‘Ae’, the observed heterozygosity ‘Ho’, and expected heterozygosity ‘He’ were 6.0, 3.6, 0.64, and 0.37, respectively. An obvious genetic deviation from Hardy–Weinberg expectation was observed both among and within cultivars and a considerable genetic variation was revealed within rather than among cultivars. It is necessary to broaden the genetic basis of the breeding germplasm in tuber mustard. Based on their geographical distributions, the tuber mustard cultivars in this study can be divided into up-Yangtze river, mid-Yangtze river, and down-Yangtze river groups. Genetic diversity was highest in mid-Yangtze river group, followed by up-Yangtze river group, and then down-Yangtze river group. It was presumed that the origin center or genetic diversity center of tuber mustard was mid-Yangtze river, and the crop was transmitted along the Yangtze river in both directions.