【Objective】This study analyzed the genetic diversity and genetic relationship of Sambucus williamsii from different provenances based on SSR molecular markers, to provide a scientific basis for the rational protection, evaluation, and utilization of S. williamsii germplasm resources.【Method】Using 245 samples of Sambucus germplasm resources and 60 samples of Sambucus hybrid germplasm as the test materials, 100 pairs of SSR primers were developed based on the transcriptome sequencing results. Through the amplification and screening of eight different individuals of Sambucus species, 13 pairs of SSR primers with good polymorphism were obtained. The genetic diversity, genetic distance, principal coordinates of different species origins, UPGMA relationships, and evolutionary trees of the Sambucus germplasm resources and their hybrid offspring were examined using the Popgene, Genemarker, GeneALeX and NTSYSpc software packages.【Result】We employed 13 pairs of fluorescent primers to amplify 305 individuals, and 162 alleles were detected. The variation range of alleles at different loci was determined as 4-34, with an average of 12.5 alleles. The total number of effective number of allele ( N_e) was 50.408, with a variation range of 1.066 1-11.070 2. The average N_e, abserved heterozygosity (H_o), and expected heterozygosity (H_e) values were 3.878, 0.331 5 and 0.521 5, respectively. Moreover, we determined the average Nei’s genetic distance (Nei) and polymorphism information content (PIC) values of 0.520 4 and 0.496 4, respectively. These results suggest a high genetic diversity. The H_o/H_e ratio at site 42 was greater than 1, indicating high degrees of heterozygosity and genetic variation within the species. Among the tested samples from different species origins, H_o ranged from 0.208 (hybrid combination 1) to 0.486 (Changbai County), with an average H_o value of 0.372. H_e ranged from 0.242 (hybrid combination 1) to 0.552 (Daqing), with an average H_e value of 0.434. Notably, the H_o/H_e ratios for the species origins (or hybrid combinations) of Changbai County, North Korea provenances, and hybrid combination 2 were all greater than 1, indicating that these three species origins (or hybrid combinations) had higher population heterozygosity and greater degrees of genetic variation within the populations. Principal coordinate analysis revealed that the majority of genetic variation, accounting for 80%, was found among individuals, while genetic variation among species origins accounted for only 20%. This may be because the cultivated varieties from different regions originated from the same provincial area, leading to smaller genetic variation among species of different origins. The genetic distance of Nei among species origins (or hybrid combinations) ranged from 0.023 to 0.637. The greatest genetic differentiation was observed between the natural populations of Yangtianshan in Qingzhou, Shandong, and Changbai County, Jilin, and the lowest between the populations of Chifeng, Inner Mongolia, and Altay, Xinjiang. The clustering could be divided into three groups, and the same source individuals were not completely clustered into the same branch, which may be caused by genetic variation or mutual introduction.【Conclusion】We demonstrated the high genetic diversity of the germplasm resources of S. williamsii. To achieve genetic improvement, intra-provenance selection and single plant improvement should initially be performed, followed by inter-provenance selection. This study provides a theoretical basis for the precise identification of S. germplasm resources, the construction of core germplasms, and the creation of new varieties.