<p>This study evaluated the effects of land-use, local environmental conditions, and the presence of non-native species on fish beta diversity in headwater streams of the Iguaçu, Piquiri, and Ivaí River basins, Brazil. A total of 25 streams were sampled over three annual campaigns. Data collection encompassed environmental variables and land-use characteristics within each stream’s drainage area. Fish were sampled using electrofishing, identified, and analyzed for species richness and abundance. For each stream, the local contribution to beta diversity was calculated and partitioned into species replacement and abundance differences. Species contribution to beta diversity were also assessed. Generalized linear models indicated that streams with greater forest cover had higher species richness in the Iguaçu and Ivaí River basins, whereas a negative relationship was observed in the Piquiri River basin, likely due to local environmental stressors. Beta diversity was driven by species replacement in preserved streams and by differential abundance in degraded streams. Non-native species made greater contributions in urbanized and agricultural areas. The research highlights the importance of conserving forested streams to sustain native fish diversity, while also recognizing that even degraded areas can contribute to variation in assemblage composition in the regional diversity. Ecological restoration of impacted streams is recommended as a conservation strategy to mitigate the effects of environmental degradation and preserve beta diversity in Neotropical stream ecosystems.</p>