ISSR markers were used to study genetic diversity and relationship of different ecotypes of longan germplasm resources. The results showed that the 12 primers screened from 100 ISSR primers and then used to amplify the genome DNAs of 39 materials. Totally 152 ISSR loci were produced, including 117 polymorphic loci, with a polymorphism rate of 76.97%. The genetic similarity coefficients among 39 different ecotypes materials of longan were between 0.57 and 0.92. Then ISSR markers can be reveal the genetic diversity of these tested materials. The cluster analysis conducted with UPGMA showed that the tropical and south subtropical ecotypes materials of longan were clustered into one major group each other based on the ISSR analysis. All of the cultivars could be separated into three groups at about 0.65 similarity value（a group of south subtropical ecotype longan from China; a group of south subtropical ecotype longan Shixia and Dawuyuan，and tropical ecotype longan Sijimi genotype; a group of longan from Vietnam and Tailand）.It was also showed that the genetic relationship was little related and has a higher genetic background between different ecotypes of longan. The results will provide much more useful information for classification, genetics and evolution of longan germplasm resources.