The Hokkaido salamander (Hynobius retardatus) is endemic to Hokkaido Island, Japan, and shows intriguing flexible phenotypic plasticity and regional morphological diversity. However, to date, allozymes and partial mitochondria DNA sequences have provided only an outline of its demographic histories and the pattern of its genetic diversification. To understand the finer details of the population structure of this species and its evolution since speciation, we genotyped five regional populations by using 12 recently developed microsatellite polymorphic markers. We found a clear population structure with low gene flow among the five populations, but a close genetic relationship between the Teshio and Kitami populations. Our demographic analysis suggested that Teshio and Erimo had the largest effective population sizes among the five populations. These findings regarding the population structure and demography of H. retardatus improve our understanding of the faunal phylogeography on Hokkaido Island and also provide fundamental genetic information that will be useful for future studies.