Dispersal is a key component of a species’ life history, by influencing population persistence, genetic structure, adaptation and maintenance of genetic diversity. The Asiatic toad (Bufo gargarizans) is a widespread species in east Asia. However, we still have no knowledge of what kind of geographical scale equates to genetic differentiation within B. gargarizans. In this study, the population genetics of B. gargarizans was studied at five localities, with the Yangtze River running through the sampling area, in order to detect the level of genetic differentiation and the natural barriers to the species’ dispersal on a small geographic scale, by means of the development and use of novel microsatellite loci. These markers revealed a relatively high level of genetic diversity. Distinct genetic structure among populations in B. gargarizans was observed, as described by genetic distance, AMOVA, PCA and Geneland results. A weak but significant positive correlation between genetic distance and geographical distance. The combination of these findings suggests that the Yangtze River and geographic distance may act as effective barriers for B. gargarizans. These results serve as benchmark data for understanding the impacts of dispersal barriers and continued landscape research on B. gargarizans.