This study determined long-term changes in fish assemblages, river discharge, salinity, and local precipitation, and examined hydrological drivers of biotic homogenization in a dryland river ecosystem, the Trans-Pecos region of the Rio Grande/Rio Bravo del Norte (USA/Mexico). Historical (1977-1989) and current (2010-2011) fish assemblages were analyzed by rarefaction analysis (species richness), nonmetric multidimensional scaling (composition/variability), multiresponse permutation procedures (composition), and paired t-test (variability). Trends in hydrological conditions (1970s-2010s) were examined by Kendall tau and quantile regression, and associations between streamfiow and specific conductance (salinity) by generalized linear models. Since the 1970s, species richness and variability of fish assemblages decreased in the Rio Grande below the confluence with the Rio Conchos (Mexico), a major tributary, but not above it. There was increased representation of lower-flow/higher-salinity tolerant species, thus making fish communities below the confluence taxonomically and functionally more homogeneous to those above it. Unlike findings elsewhere, this biotic homogenization was due primarily to changes in the relative abundances of native species. While Rio Conchos discharge was > 2-fold higher than Rio Grande discharge above their confluence, Rio Conchos discharge decreased during the study period causing Rio Grande discharge below the confluence to also decrease. Rio Conchos salinity is lower than Rio Grande salinity above their confluence and, as Rio Conchos discharge decreased, it caused Rio Grande salinity below the confluence to increase (reduced dilution). Trends in discharge did not correspond to trends in precipitation except at extreme-high (90th quantile) levels. In conclusion, decreasing discharge from the Rio Conchos has led to decreasing flow and increasing salinity in the Rio Grande below the confluence. This spatially uneven desertification and salinization of the Rio Grande has in turn led to a region-wide homogenization of hydrological conditions and of taxonomic and functional attributes of fish assemblages.