For genetic analysis of persimmon (Diospyros kaki) and its relatives, we investigated the development of SSR markers with expression sequence tags (ESTs). Of 9475 EST data from the NCBI database which had isolated from D. kaki, 746 sequences contained SSRs which had more than four (tetranucleotide) or five (dinucleotide and trinucleotide) repeats. Tested on two species, D. kaki and D. lotus (a diploid relative of D. kaki), of primer pairs based on 65 SSR-containing EST sequences, 52 primer pairs resulted in proper PCR products. Some of these candidate EST-SSR markers showed polymorphism among cultivars and seedlings of D. kaki and D. lotus. Among tested seven clones of D. lotus, 32 of 52 candidate EST-SSR markers showed polymorphism. For molecular phylogenetic analysis, linkage mapping and whole genome mapping of Diospyros species, EST-SSR markers appear to be as useful as genomic SSR markers. In addition, development of EST-SSR markers seems to be easy and cost-effective when compared with genomic SSR markers.