Biodiversity is responsible for important ecological processes like productivity and ecosystem stability, and rare species are a major component of biodiversity. Rarity increases a species' vulnerability to disturbances and also makes them difficult to study. Globally, species of freshwater systems are some of the most threatened, and evaluation of rare freshwater species and their habitats is needed to help preserve natural flexibility and ecological function. We conducted an analysis of full fish communities of the upper St. Lawrence River and its major US tributaries, with the goals of determining species locations and abundances, associated environmental conditions, the distribution of distinct fish assemblages across the landscape (with emphasis on communities supporting rare species), and potential threats. From 2009 to 2015, the US Geological Survey (USGS) and Saint Regis Mohawk Tribe (SRMT) worked together using standardized methods to collect community samples within 4 different aquatic realms (shallow and deep lentic, and small and large lotic systems) and determine species-specific fish abundances, frequencies of occurrence, and associated habitat signatures and spatial distributions. Distinct fish assemblages and associated habitat conditions were objectively identified by multivariate and hypothesis-testing methods. We used a geographic information system (GIS) to spatially associate habitat, biotic, and landscape attributes within each stream reach throughout the study area, facilitating quantification of distribution patterns. Comparisons with historical data provided estimates of loss or gain of threatened and endangered species (T&E) colonies. We developed a disturbance index to highlight potential threats to aquatic species. More than 140,000 fishes of 87 species were collected from a total of 1140 sample sites, covering 278 stream reaches, including the endangered Notropis anogenus (Pugnose Shiner), and threatened Hiodon tergisus (Mooneye), Etheostoma pellucidum (Eastern Sand Darter), and Acipenser fulvescens (Lake Sturgeon). We identified 50 distinct fish assemblages differing in species composition, abundance, and/or diversity, but only 13 of those assemblages included a T&E species. The rareness, extent, and patchiness of fish assemblages created a mosaic of fish communities across the landscape, from headwaters to the mainstem of the St. Lawrence River. Comparisons with historic surveys (1978–2008) showed a stable number of T&E species colonies or an increase for some species. The geographic distribution of multimetric disturbance index values showed where combinations of disturbances to fish habitats might affect rare fish species and aquatic communities in the region. The species–habitat associations and fish assemblage distributions can be used for evaluation of species, communities, or habitats that may need protection or restoration.