Landscape features, such as rivers, can act as geographical barriers to dispersal and gene flow and thus influence the population structure of some species. In this study, tissue samples were collected from 73 Hume's Pheasant Syrmaticus humiae, from six localities in Guangxi and Guizhou provinces, China, to examine the influence of rivers in landscape structure on genetic diversity and structure based on 12 microsatellite loci. Results indicated a high genetic diversity in Hume's Pheasant. Individuals from populations in Tianlin, Longlin and Xilin counties (TXL) (three geographically proximate populations) tended to form a genetic cluster, distinct from three other geographically proximate populations 100 km to the west in Pojie town (PJ), Luodian county (LD) and Leye county (LX), which showed more mixing and were less genetically distinct. Using simulated Markov-switching VAR (MSVAR), we found that the median population sizes of the posterior distributions were approximately 3,715 individuals for N₀, and approximately 100,000 for N₁, indicating that Hume's Pheasant experienced a significant genetic bottleneck 4,800 years ago, possibly due to human activity. Hume's Pheasant shows female-biased dispersal. The results of STRUCTURE and GENELAND indicate that Nanpan River, Hongshui River and national road G324 act as potential genetic barriers for Hume's Pheasant in Guangxi and Guizhou provinces. In addition, genetic distinctiveness has persisted despite population declines of the Hume's Pheasant due to the bottleneck approximately 5,000 years ago and population declines in the last 100 years.