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Rape (Brassica napus) is an important oilseed crop widely cultivated worldwide. Due to its relatively short evolutionary and domestication history, its intra-species genetic diversity is limited. Radish (Raphanus sativus), belonging to a different genus but the same family as B. nupus, possesses an abundance of excellent gene resources. It is commonly used for B. nupus germplasm improvement and genetic basis expansion, making it one of the most important close relatives for distant hybridization. In the present study, a novel method for detecting alien chromosome fragments, called Anchor Mapping of Alien Chromosome (AMAC) was used to identify radish chromosome segments in the progeny of rape–radish interspecific hybrids. Based on the AMAC method, 126,861 pairs of IP (Intron Polymorphism) and 76,764 pairs of SSR (Simple Sequence Repeat) primers were developed using the radish Rs1.0 reference genome. A total of 44,176 markers (23,816 pairs of IP and 20,360 pairs of SSR markers) were predicted to be radish genome specific-single-locus (SSL) markers through electronic PCR analysis among four R. sativus, one B. napus, one B. rapa, one B. juncea, and one B. juncea reference genome. Among them, 626 randomly synthesized SSL markers (478 SSL IP markers and 148 SSL SSR markers) were used to amplify the genome of 24 radish samples (R. sativus), 18 rape (B. napus), 2 Chinese cabbage (B. rapa), 2 kale (B. oleracea), and 2 mustard (B. juncea) samples, respectively. Then, 333 SSL markers of the radish genome were identified, which only amplified in the radish genome and not in any Brassica species genome, including 192 IP markers and 141 SSR markers. Furthermore, these validated SSL markers were used to identify alien chromosome fragments in Ogura-CMS restorer line 16C, Ogura-CMS sterile line 81A, and their hybrid-Yunyouza15. In 16C, one marker, Rs1.0025823_intron_3, had an amplification product designated as anchor marker for the alien chromosome fragment of 16C. Afterwards, four novel radish genome-specific IP markers were found to be flanking the anchor marker, and it was determined that the alien chromosome segment in 16C originated from the region 8.4807–11.7798 Mb on radish chromosome R9, and it was approximately 3.2991 Mb in size. These results demonstrate that the AMAC method developed in this study is efficient, convenient, and cost-effective for identifying excellent alien chromosome fragments/genes in distant hybrid progeny, and it can be applied to the molecular marker-assisted breeding and hybrid identification of radish and Brassica crop species. [ABSTRACT FROM AUTHOR]