The history of chestnut utilization in Japan is very long. Many cultivars have been preserved without being sufficiently organized and studied with respect to genetic variation. We developed 12 new SSR (simple sequence repeat) markers from Japanese chestnut (Castanea crenata) by using SSR-enriched genomic libraries. All 12 primer pairs selected for SSR analysis successfully amplified fragments in 6 Castanea species. The genetic diversity and relationships among 216 chestnut accessions were assessed by SSR markers, including 142 Japanese chestnuts, 38 Chinese chestnuts (C. mollissima), 2 European chestnuts (C. sativa), 9 American chestnuts (C. dentata), and 23 Japanese–Chinese hybrids. Among the 216 accessions, we identified 21 synonym groups (including 1 bud sport group), and 189 different genotypes. The mean values of observed heterozygosity (H_O) and the expected heterozygosity (H_E) in the Japanese chestnuts were 0.65 and 0.65, respectively. A phenogram of the 189 genotypes revealed 3 major groups, corresponding to Japanese, Chinese and American chestnuts. Japanese–Chinese hybrid cultivars were mainly included in the Chinese chestnut group. In the present study, genetic backgrounds could be identified for native and cultivated Japanese chestnuts and Japanese–Chinese hybrid cultivars, and many synonyms showing identical SSR genotypes were found. Genetic and geographical relationships of unique characteristics such as “Shidareguri” (weeping) and “Togenashi” (spineless) were also discussed. These results will improve the efficiency of cultivar identification and of chestnut breeding programs.