Compared with other terrestrial environments, the stream environment generally presents a linear spatial structure and relatively simple environment. In a stream landscape, the dispersal direction of stream-type organisms usually presents a linear structure along the stream, which results in the limited dispersal and the genetic differentiation of stream-type organisms across different stream sections. The Shangcheng Stout Salamander (Pachyhynobius shangchengensis) is a narrowly distributed stream salamander in Dabie Mountains of East China. In the present study, we tested for the impact of stream landscape (i.e. waterfalls and underground river) on genetic structure and dispersal patt in P. shangchengensis based on12 nuclear microsatellite loci from195 individuals in 3 populations (A, B and C) from three closely connected sections within one stream. Genetic diversity results suggested that Population B contains relatively high genetic diversity for P. shangchengensis when compared to the other populations (A and C). Detectable genetic differentiation was found (FST = 0.008, P = 0.007) among three populations, which was also supported by the Structure, FCA analysis and relatedness estimates of each pair of individuals among populations. The assignment test suggested that P. shangchengensis has philopatric males and female-biased dispersal (mean female Alc = ?0.031, SE= 0.225; mean male Alc = 0.026, SE = 0.198). Female-biased dispersal was also supported by analyses for each sex (i.e. Spatial autocorrelation, Genetic distance, Relatedness analysis). Our study indicated that small and isolated populations (A and C) had relatively low genetic diversity due to the limited population size. For stream salamanders, landscape features (i.e. waterfalls and underground river) can influence the ability of an individual todisperse through the landscape, and consequently influence the formation of strong genetic differentiation of P.shangchengensis.