In river ecosystems, spatial complexity as well as anthropogenic factors operating at different temporal and spatial scales are shaping demography, connectivity and population genetic structure of species inhabiting these habitats. Chilina dombeiana is a freshwater gastropod with direct development (absence of a free larval phase) that inhabits the Biobío river basin in Chile (36°S). No studies have yet evaluated the spatial patterns of the genetic diversity of this species and the potential factors that influence these patterns. Consequently, in this study, we analyzed the population genetics of C. dombeiana based on 15 locations along the Biobío river. Eight microsatellite loci were genotyped. Also, at each sampling site, 40 environmental parameters were recorded to characterize them. Results showed that C. dombeiana has low genetic variability with high population structure. In addition, we detected signs of historical decreases in effective population sizes, unidirectional gene flow (upstream to downstream) and contemporary demographic bottleneck. Spatial subdivisions in populations showed a pattern of isolation by distance. The redundancy analysis and variance partitioning showed that spatial components and dissolved oxygen could explain 28% of the interpopulation genetic variation, while the Random Forest analysis identified significant effects of dissolved oxygen, nitrite and total coliforms on the genetic variability of populations (22%). Although C. dombeiana is widely distributed in Chilean rivers, its low dispersal and specific habitat requirements make this species very sensitive to the severe increase in anthropogenic disturbances affecting river ecosystems in recent decades. Long-term monitoring of genetic population conditions and environmental parameters are needed to implement robust management and conservation policies.