Chimonanthus praecox (wintersweet) is endemic to China. It has been cultivated there for more than 1000 years as a garden, potted, and cut-flower plant. Many cultivars have been developed during its long history of cultivation, and recently many germplasms were collected in Wuhan and Nanjing, China. The identification and genetic relationship of these resources were studied based mainly on morphological traits. In the current study, intersimple sequence repeat markers (ISSR) and random amplified polymorphic DNA markers (RAPD) were used for the first time to investigate 72 wintersweet clones from the two regions. Eleven ISSR primers amplified 115 bands, 90 (78.26%) of which were polymorphic. Nineteen RAPD primers amplified 165 bands, 105 (63.63%) of which were polymorphic. Either ISSR or RAPD markers were sufficient to distinguish all the clones surveyed. A Dendrogram based on Jaccard's similarity coefficients indicated that the distribution pattern of the 72 clones was coherent with their geographical origins. Most of the genetic variation (85.68% with ISSR data; 86.75% with RAPD data) occurred among clones within each region. However, the difference between Wuhan and Nanjing groups is statistically significant (ΦST = 0.143, P < 0.001, with ISSR data; ΦST = 0.132, P < 0.001, with RAPD data). Morphological variation and classification of wintersweet cultivars were also discussed compared with the genetic relationship based on ISSR and RAPD markers. This is the first report of the partitioning of genetic variability within and between different cultivated wintersweet regions, and it provides useful baseline data for optimizing sampling strategies in breeding. These results are important for future genetic improvement, identification, and conservation of Chimonanthus praecox germplasm.