The Mammoth Spring crayfish (Orconectes marchandi) is listed as endangered by the American Fisheries Society's Endangered Species Committee and globally impaired by the Missouri Natural Heritage Database. Recorded at only three locations in the Spring River watershed (southern Missouri and northern Arkansas), little information exists on its range, associations with other crayfish and habitat selection. We sampled stream-dwelling crayfish with kicknets and quadrat samplers during spring-summer over 2 y in the Spring River drainage to determine distribution, relative density, habitat use, species associations, and life history characteristics of O. marchandi and other associated crayfish species. Nine crayfish species were collected in the Spring River watershed including a previously unrecorded invasive species, O. neglectus chaenodactylus, that appears to have replaced the native O. eupunctus from a portion of the watershed where it was previously abundant. The known distribution of O. marchandi was expanded from three streams in three sub-watersheds to 20 streams in eight sub-watersheds. Orconectes marchandi was found mainly in smaller streams (order 1–3) in habitats consisting of slower moving, shallow water with gravel, pebble and cobble substrates. Oronectes marchandi was not significantly associated with any crayfish species by site. Orconectes eupunctus was found exclusively in the main-channel South Fork and Spring rivers. Current velocity, water depth and substrate were related to crayfish densities, but were crayfish species- and size-dependent. Growth rates differed among species with O. punctimanus showing the greatest growth in carapace length from winter to summer and O. marchandi showing the greatest increase in weight with carapace length. Sex ratios differed among crayfish species and by season. Additional research is needed to gain a better understanding of factors affecting habitat selection by crayfish species and to determine potential impacts of O. neglectus chaenodactylus on native populations.