We investigated the genetic structure of 16 populations (from five geographical groups) of clouded salamander (Hynobius nebulosus) in northern Kyushu, Japan, by analyzing the sequence of the mitochondrial DNA control region. A total of 24 haplotypes were found in 199 individuals of the salamander. In the analysis of genetic diversity of the populations, an isolated small population sustained in an urban area had a low level of genetic diversity. This is considered to be due to both the less suitable nature of the habitat and a recent artificial barrier to gene flow. In the analysis of genetic differentiation (F_ST) among 16 populations, significant differences were detected in 76.7% of all possible population pairs. Analysis of molecular variance (AMOVA) showed that most of the genetic variation was attributed to individual differences within populations, but a significant genetic difference among groups was also detected. This fine-scale genetic differentiation would be formed through genetic isolation (or gene flow) by geographic distance, because strong correlations between genetic F_ST/(1-F_ST) and geographic distances were detected. In 10 northern populations from two adjacent groups that were separated by a small lowland river there is a significant positive correlation between geographic and genetic distance, but there tends to be a larger genetic distance between the inter-group populations than the distance within groups. These results suggest that even a small lowland river, if combined with past rising of sea level by transgression, might be a landscape barrier to gene flow of this species.