Background: Pervasive introductions of non-native taxa are behind processes of homogenization of various types affecting the global flora and fauna. Chile’s freshwater ecosystems encompass a diverse and highly endemic fish fauna that might be sensitive to the introduction of non-native species, an ongoing process that started two centuries ago, but has to date received little attention. Using historical (native) and present-day (native and non-native) presence-absence data sets of compositional similarity, our goal was twofold: (1) evaluate patterns of taxonomic homogenization at various spatial scales and (2) identify clusters of widely versus narrowly distributed species to assess their relative role in compositional changes. We expect that non-native species with wide distributions might have a larger influence in taxonomic homogenization than those with narrow distributions. Results: Chile’s fish assemblages have become increasingly homogenized during the last two centuries when evaluating changes in compositional similarity among 201 watersheds (65.3 % of total comparisons showed homogenization) distributed among six defined biotic units. Taxonomic differentiation was significantly more prevalent than taxonomic homogenization within biotic units. Among biotic units, comparisons between historical and current compositional similarity were all significantly different. We identified one cluster of non-native fishes that were distributed across the entire five or six biotic units. This cluster included Brown Trout (Salmo trutta) and Rainbow Trout (Oncorhynchus mykiss) as the two most representative species. A second cluster we identified included fishes such that on average spanned only one or two biotic units. Conclusions: We provide first evidence for an ongoing and large-scale process of taxonomic homogenization among Chile’s watersheds occurring at various scales. Our findings provide taxonomic and biogeographic baseline information for management plans and courses of action for conservation of native fishes, many of which are endemic. We also discuss management guidelines of non-native fishes in Chile. Baseline information of both native and non-native fish taxa might be applicable to other isolated regions elsewhere.