To evaluate the genetic diversity and to clarify the genetic relationships of Japanese peach cultivars, we analyzed the amplified fragment length polymorphism (AFLP) and traced the pedigree of 17 Japanese commercial peach cultivars and six traditional accessions. Sixteen AFLP primer combinations produced a total of 837 fragments and 146 polymorphic bands with a polymorphism percentage of 17.5%. All of the peach accessions could be identified from differences in at least 10 polymorphic bands. A cluster analysis showed that all the Japanese commercial peach cultivars, except ‘Kiyomi’ and ‘Jichigetsuto’, formed a major group consisting of three sub-groups. Of the six traditional accessions, four were genetically distant from the Japanese commercial peach cultivars while two accessions from China were classified into the Japanese commercial peach cultivars group. Both the AFLP analysis and pedigree tracing suggested that Japanese commercial peach cultivars are mainly derived from ‘Shanhai Suimitsuto’, one of the traditional accessions from China. Although the genetic relationships revealed by AFLP were generally in agreement with those shown by the pedigree information, some contradictions were found. Combining the AFLP results and pedigree information can provide a better understanding of the genetic relationships of Japanese peach cultivars.