A new Brassica napus cytoplasmic male sterile (CMS) line, ‘93-12A’ was developed through cross and continuous backcross between tuber mustard CMS line and the Brassica napus variety ‘92-58’. By backcrossing it to the inbred lines ‘022058’, we finally got a stable CMS line named ‘Jiayou58A’ with improved comprehensive agronomic characters. To identify the origin of cytoplasmic male sterility of ‘93-12A’ and ‘Jiayou58A’, the specific primers were amplified according to the reported fertility-related gene orf224, orf138 of B. napus and orf220 of B. juncea in its genome. The results showed that only the specific band of orf220 was detected in the new CMS line ‘93-12A’ and the improved CMS line ‘Jiayou58A’. Sequencing of the bands confirmed that the male sterility cytoplasm of ‘93-12A’ and ‘Jiayou58A’ is originated from the tuber mustard (B. juncea).