Brassica juncea is an important specialty vegetable and processing material in China, used for fresh consumption, processing, seasoning, and oil extraction. Among these, the processing-type mustard industry has a large scale and irreplaceable economic value. Turnip mosaic virus (TuMV), as the most damaging viral disease in mustard, severely restricts mustard production, causing stunting, chlorosis, and significant reductions in yield and commercial quality, thus constraining the high-quality and sustainable development of the vegetable industry. In this study, 205 mustard germplasm accessions were inoculated by mechanical rubbing at the seedling stage and subjected to ELISA to evaluate their resistance to TuMV. The results showed significant differences in resistance among the 205 accessions, with disease index ranging from 0 to 100. A total of 3 immune, 16 highly resistant, and 4 resistant accessions were screened out. Using incidence rate and disease index as variables, the genetic distance between accessions was calculated based on squared Euclidean distance. Systematic cluster analysis using the between-groups linkage method classified the materials into five groups, which showed high consistency with the resistance grading results. Further ELISA analysis of 83 representative Brassica juncea accessions revealed that the phenotypes were largely consistent with virus accumulation levels. The resistant germplasm mainly originated from Fujian, Sichuan, and Guangxi provinces, with predominant varieties being Brassica juncea var. crassicaulis Chen et Yang, Brassica juncea var. megarrhiza Tsen et Lee, and Brassica juncea var. Latipa Li. The elite resistant sources identified in this study provide a material basis for TuMV-resistant breeding and new variety development in Brassica juncea.