Abstract The genetic diversity of 31 germplasm resources of Myrica rubra from Zhejiang Province, the major gathering site and the largest producer of M. rubra in China, was investigated using inter-primer binding site (iPBS) and start codon-targeted polymorphism (SCoT) markers. Cluster analysis was performed using unweighted pair-group method arithmetic averages. The results showed that the genetic similarity (GS) values of the samples tested using the two molecular markers were within the range of 0.5 to 0.94. Approximately 452 distinct bands, including 401 polymorphic bands, were obtained using 38 pairs of primers, and the polymorphism rate was 88.72%. A threshold GS value of 0.70 was used to divide the 31 M. rubra samples into three groups. The four major M. rubra varieties in China that originated from Zhejiang Province (Dongkui, Biqi, Dingaomei, and Zhoushanwandaomei) showed significant genetic differences. Dongkui was more distantly related to Dingaomei and Zhoushanwandaomei compared to Biqi. The GS values of the Dongkui mutant samples DB1 and DB2 were 0.77 and 0.80, respectively, and thus they were considered to be more closely related. The GS values of Biqi and its bud mutants Zaoqi, Yongxuan 56, and Wuzi were 0.66, 0.59, 0.658, and 0.65, respectively, with Zaoqi showing more differences than the other three varieties. Zhoushanwandaomei and Shangyubaiyangmei, Heijing and Wuzi, Yongjiacimei and Yuanqiaoshuimei, Dongaowumei and Jiangkou Dongkui monoecious plants, Tuda and Liuyemei, Xuemei and Haochimei, Donglingwubian and Muyemei, DB2 and Dongkuimushu, and Dongkui and Shanzhan Dongkui monoecious plants were all clustered within the same subgroup, indicating that these were more closely related. The GS of both Xiyegaozhuang and Jiazhaizao was 0.94, suggesting that these varieties were synonymous. The GS of Linhaiyangpingmei was 0.55, indicating that it had the most distant genetic relationship. For the three major resource-gathering sites – Taizhou, Ningbo, and Wenzhou – the intra-regional genetic germplasm base was narrow with relatively extensive exchanges, and the sites were extra-regionally independent. Our study results are consistent with those of morphological index analysis, have identified the conditions in which the genetic diversity of M. rubra germplasm in Zhejiang Province occurs, and have established a foundation for genetic breeding and germplasm research and identification.