The morphological characteristics and genetic relationships among 32 germplasms of Zanthoxylum schinifolium and Zanthoxylum piperitum collected from two farms in Korea were investigated. The traits with the most variability were seed color, leaf size, and spine size. The intraspecific polymorphism of Z. schinifolium and Z. piperitum was 96.5% and 60.3%, respectively. The genetic diversity and Shannon’s information index values ranged from 0.11 to 0.33 and 0.19 to 0.50, with average values of 0.26 and 0.42, respectively. Two ISSR primers (UBC861 and UBC862) were able to distinguish the different species. The genetic similarity matrix (GSM) revealed variability among the accessions ranging from 0.116 to 0.816. The intraspecific GSM for Z. schinifolium and Z. piperitum was 0.177–0.780 and 0.250–0.816, respectively. The GSM findings indicate that Z. schinifolium and Z. piperitum accessions have high genetic diversity and possess germplasms qualifying as good genetic resources for cross breeding. The clustering analysis separated Z. schinifolium and Z. piperitum into independent groups, and all accessions could be classified into three categories. Z. Schinifolium var. nermis belonged to independent groups. Comparison of the clusters based on morphological analysis with those based on ISSR data resulted in an unclear pattern of division among the accessions. The study findings indicate that Z. schinifolium and Z. piperitum accessions have genetic diversity, and ISSR markers were useful for identifying Z. schinifolium and Z. piperitum.