【Objective】The present study explored the genetic diversity and relationships of Guangxi ancient litchi germplasm resources  in order to provide references for utilization of germplasm resources and variety breeding. 【Method】The genetic relationships of 24 ancient litchi varieties were analyzed using ISSR molecular marker. 【Result】Thirteen primers with clear bands and favorable repeatability were selected from 100 ISSR primers； 96 bands were amplified altogether and 43 of them were polymorphic bands with a polymorphism proportion of 44.79%， and 7.4 bands were amplified by each primer on average. The genetic similarity coefficients among the tested ancient litchi germplasm resources ranged from 0.43 to 1.00 with average relative genetic similarity coefficient of 0.61. UPGMA cluster analysis results indicated that， the 24 tested germplasm resources of ancient litchi could be classified into three groups when genetic similarity coefficient was 0.66. Group Ⅰ came from Lingshan county， Guangxi； Group Ⅱ derived from Guiping city， Beiliu cuty, Yulin city, Lingshan county and Daxin county in Guangxi； Group Ⅲ came from Beiliu city and Lingshan county in Guangxi. 【Conclusion】The hereditary basis among the 24 varieties of ancient litchi in Guangxi is extensive， and they can serve as excellent materials for cross breeding of litchi in the future.