Abstract: In order to understand the genetic diversity of ancient tea trees in Sui Autonomous County of Sandu of Guizhou province, the ISSR markers were used to analyze the genetic diversity and genetic relationships of 145 ancient tea trees. And, the results showed a total of 15 primers with good polymorphism, repeatability and clear amplification were screened from 100 ISSR universal primers. The selected primers were used to amplify the genomic DNA of the tested ancient tea trees. Meanwhile, a total of 116 bands were amplified by 15 primers, of which 110 were polymorphic, and the average ratio of polymorphic bands was 94.37%. The average number of alleles（Na）, the average number of effective alleles（Ne） and the average Nei’s genetic diversity（He） were 1.945 7, 1.529 3 and 0.312 6 respectively for 145 ancient tea trees calculated by Popgene 32. The average Shannon information diversity index（I） was 0.471 2, and the genetic consistency ranged from 0.448 3 to 0.965 5. Further,The NTSYS-pc 2.1 was used to construct UPGMA cluster diagram.The 145 samples of ancient tea trees were divided into four groups at the similarity coefficient of 0.72,and the clustering results were consistent with the results of morphological clustering.This study provided a theoretical basis for the protection,utilization and cultivation of ancient tea germplasm resources.