Restriction fragment mapping of mitochondrial DNA (mtDNA) was used to examine the genetic relationships among several populations of wrinkled frog, Rana rugosa, of which are among the largest yet reported in vertebrate species. The analysis of restriction pattern and the mitochondrial genome sizes revealed two distinct mtDNA groups, deriving from either addition or deletion of large or small mtDNA fragments. The mean percent of the sequence divergence between the two groups was high, estimated to be 8.20%, ranging from 2.90 to 12.40%, based on the restriction fragment data. The degree of difference between the two groups was much higher than that observed in other species. Based on these data, R. rugosa mtDNA may be divided into Eastern and Western groups. However, there is on e population (Donghae) which shows the mtDNA characteristics of both groups. Although the size variation of mtDNA is often observed in amphibians, such large differences of mtDNA in R. rugosa are considered to be a genetic variation rarely found.