Freshwater streams and rivers exist as complex networks of waterways that intersect the landscape. While some degree of natural isolation occurs, e.g. headwater streams, in general freshwater taxa have evolved in a relatively connected riverscape where longitudinal and lateral dispersal are possible. However, anthropogenic environmental changes, such as the construction of dams and weirs, have disrupted the natural dispersal potential of many freshwater taxa. The overall aim of this study was to assess how loss of connectivity due to small weirs effects fish abundance, community composition, size structure, taxa richness, genetic diversity, and genetic structure at a local scale. Our study area was an approximately 60km2 area around Ueda city, Nagano Prefecture that included the Chikuma River mainstream (3 sites) and several small natural and semi-natural tributaries (15 sites). We assessed fragmentation in the survey area by searching for small barriers on satellite imagery followed by in site inspections. To assess the fish community, we performed eDNA and electrofishing surveys in Sept 2022 and June 2023. We also assessed habitat heterogeneity and sampled macroinvertebrates as bioindicators of water quality following the Ministry of Environment protocol. Finally, we performed a riverscape genetic analysis of a target species, Amur minnow (aburahaya). The results indicated that overall taxa diversity and genetic diversity decreased in locations that were naturally isolated (further away from the mainstream) and more fragmented (larger number of barriers between the site and mainstream). Genetic structure of the fish community, and specifically Amur minnow, was also positively associated with isolation and number of barriers. Our study highlights the importance of considering connectivity in freshwater ecosystems, even at the local mainstream-tributary scale.