This study attempted to establish a high-throughput and rapid method using SSR markers, in order to clarify the mustard varieties (Brassica juncea L.) released from China. Sixteen representative mustard varieties were used to screen SSR primers showing polymorphism. Out of 432 pairs of tested primers, 84 were detected with higher polymorphism and good repeatability, and they were subjected for synthesizing the fluorescent-labeled primers. We further genotyped 96 varieties with updated versions of primers using gene analyzer. Based on the amplification stability, the readability of peaks, polymorphism and chromosome location, etc., 25 SSR markers were finally qualified. We further provided the solution using reference varieties to eliminate genotyping errors caused by different experimental batches or platforms. By taking use of these markers from six groups according to the fluorescence color and amplification fragment size, a SSR-based mustard variety identification system has been established. The DNA database containing the fingerprinting information of 189 mustard varieties was generated showing a total of 175 allelic variants with an average of 7 allelic variants per primer. The gene diversity index was variable from 0.239 to 0.870, with an average of 0.555, while the polymorphism was observed between 0.23 and 0.86, with an average of 0.493. Collectively, this study established a mustard variety clarification method by taking use of 25 SSR markers on the capillary electrophoresis genotyping platform, which might be useful in rapid authenticity identification, genetic diversity analysis and similar varieties selection in DUS test in mustard.