Chloroplast inter-simple sequence repeat markers in mango were developed and used to analyze the genetic relationship and diversity of mango and its relatives. Thirty-six mango cultivars (Mangifera indica L.) and its relative species collected from the fruit germplasm collection in the Guangxi Academy of Agricultural Sciences, China, were examined by ISSR-PCR with chloroplast DNA (cpDNA). Eight better primers for chloroplast DNA that provided reproducible, polymorphic DNA amplification patterns were screened from 50 ISSR primers and used for UPGMA analysis. According to the band patterns with 8 primers for chloroplast DNA, all cultivars tested were distinguished from each other and these showed ample genetic diversity; the average percentage of polymorphism was 77.2%. The 36 samples could be clustered into four groups by UPGMA analysis at the coefficient 0.74. The results indicated that the cpISSR marker was a new powerful tool for the identification of mango cultivars or its relative species, and their genetic relationship analysis and diversity evaluation. Chloroplast inter-simple sequence repeat markers in mango were developed and used to analyze the genetic relationship and diversity of mango and its relatives. Thirty-six mango cultivars (Mangifera indica L.) and its relative species collected from the fruit germplasm collection in the Guangxi Academy of Agricultural Sciences, China, were examined by ISSR-PCR with chloroplast DNA (cpDNA). Eight better primers for chloroplast DNA that provided reproducible, polymorphic DNA amplification patterns were screened from 50 ISSR primers and used for UPGMA analysis. According to the band patterns with 8 primers for chloroplast DNA, all cultivars tested were distinguished from each other and these showed ample genetic diversity; the average percentage of polymorphism was 77.2%. The 36 samples could be clustered into four groups by UPGMA analysis at the coefficient 0.74. The results indicated that the cpISSR marker was a new forking tool for the identification of mango cultivars or its relative species, and their genetic relationship analysis and diversity evaluation.