A large number of old tea plant resource is widely distributed in Jiuan city of Guizhou province, China. The genetic relationship of these tea plant resources still remain unclear. In this study, one-hundred old tea plant samples, which were collected from five different regions of Jiuan city, were subjected for diversity analysis by genotyping by sequencing (GBS). A total of 548,597 high-quality SNPs were identified in this collection, and the variation of these SNPs were annotated. By using these genome-wide SNPs, the phylogenetic tree, the principal component analysis (PCA) and population structure were conducted. A similar pattern on genetic relationship was revealed by three approaches. These samples were assigned into three groups and the samples collected in the same/similar area were clustered in each group. Thus, our results suggested a genetic difference of samples in the Guchayuan (G), relative to the samples from other four regions, which likely shared the same and unique parental origin.