Los Terreros Creek, a reservoir-fragmented tributary in the upper Lerma River system of central Mexico, was sampled at four localities in wet (2001) and dry (2002) seasons to evaluate spatial and temporal patterns of fish assemblages, the conservation status of native fishes, and prevalence of exotic species. Two of five species present were non-native; the common carp (Cyprinus carpio) and the shortfin silverside (Chirostoma humboldtianum). Fish assemblages formed a strong gradient largely separating the lentic, reservoir-impacted, locality (downstream-most site) from the remaining lotic sites. The two most-upstream sites were not distinguishable based on structure of fish assemblages. Three of five species showed significant spatial variation in abundance. The Aztec shiner (Aztecula sallaei) occurred only at the lotic sites and the two species of silversides (Chirostoma) occurred only at the lentic site. Temporal trends in assemblages were less evident, but assemblages in the dry season had lower mean species-richness and mean abundance values. Analysis of distance matrices based on assemblages, site-specific environmental conditions, and spatial structure indicated that a strong, spatially structured environmental gradient was significantly associated with fish assemblages in the wet season, but not assemblages in the dry season. Los Terreros Creek still supports an assemblage of native fish that is typical for the upper Lerma River system. However, this relict system remains threatened by anthropogenic activities including changes in land use and further introduction of exotic species.