The influence of habitat fragmentation on population genetic structure is among key concerns in conservation biology, particularly in the context of the Anthropocene. While extensive studies have focused on the genetic consequences of physical barriers, the influence of multiple types of barriers including thermal barriers (the presence of unsuitable thermal habitat) remains underexplored. The genus Cottus (family Cottidae), commonly known as sculpins, serves as a pertinent example of sedentary cold-stenothermic fish inhabiting the Toyohira River in Hokkaido. Our previous research documented a marked decline in their abundance in the bypassed reach with significantly elevated water temperatures, resulting from the flow diversion of a run-of-the-river dam. This study aims to elucidate the influence of the thermal barrier on the genetic structure of Wrinklehead sculpin (Cottus nozawae) by comparing it with other types of barriers. We sampled 495 individuals from 22 populations across approximately 40 km of stream networks. Caudal fins were collected and genotyped at seven microsatellite loci commonly used in studies of the Cottus genus. Our results revealed distinct dominated clusters in three regions, indicating significant genetic structure among populations at the research site. While the thermal barrier was not identified as a strong driver of the genetic differentiation, the role of a physical barrier (natural waterfall) was emphasized instead. Additionally, landscape features such as dams, gorges, and springs also potentially contributed to population isolation. The populations in the region without major physical barriers extending to a tributary exhibits significantly higher allelic richness than the other regions characterized by physical barriers. Regional differences account for approximately 4% of the detected molecular variance, which is higher than the 1% of variance among populations. Our findings highlight the distinct roles of thermal and physical barriers in shaping genetic structure within riverine ecosystems. While thermal barriers may not currently represent a major ecological conservation concern in the Toyohira River, likely due to their existence duration and/or their seasonal variability, management strategies for physical barriers are crucial to preventing the synergistic negative effects of habitat fragmentation and large-scale environmental changes, such as global warming.