We sampled fishes with a small trawl in the deep midchannel and with beach seines on nearby sandy beaches at five sites along two parallel headwater tributaries of the upper Amazon in Eastern Ecuador. We quantified ecological distance between fish assemblages of deep-river and beach habitats and compared assemblage composition, species richness, and diversity. Results of detrended correspondence analyses (DCA) and two-way indicator species analyses (TWINSPAN) clearly revealed the presence of a striking faunal change (i.e., about 92% dissimilarity) between beach-zone and off-shore fishes. The boundary between these assemblages occurred at about 2 m depth. Beach samples collected as far apart as 325 km were more similar to each other than to trawl samples taken only 10s of meters away. The beach-zone fish assemblage was strongly dominated by small characins, whereas the deep-river habitat was dominated by catfishes and weakly electric gymnotiforms. Apparent adaptations to deep-water habitats included small size, flattened or elongated body, ventral mouths, reduced eyes, and sensory specializations for life in what may be a nearly light-less environment (e.g., chemo- and electroreceptors). Visually oriented fishes and those dependent on algal or detrital foods were mostly absent from the deep-river samples. We caught many more species along the beaches, but after correcting for higher numbers of individuals collected in beach habitats, species richness was not noticeably different between the two habitats. Species diversity also averaged slightly higher for beach samples, but the difference was not significant. We infer that the deep-river habitat was undersampled and that further effort in the deep river would reveal the presence of many more species.