A history of overexploitation and industrialization of riverine habitats has impacted the Shortnose Sturgeon Acipenser brevirostrum, leading this species to become one of the earliest listed under the U.S. Endangered Species Act. The present understanding of Shortnose Sturgeon spatial ecology is based on observations from a limited number of Atlantic coastal rivers. To better understand Shortnose Sturgeon in the Hudson River, New York, we used acoustic telemetry to characterize seasonal habitat use and to identify regions of the river where seasonal sturgeon activity occurred. From 2012 to 2016, 101 adult fish were tagged and tracked, and sturgeon detections per unit effort (a metric of fish observation standardized by search effort) were evaluated against benthic habitat variables by using generalized additive regression models. Models indicated strong habitat associations in the spring season defined by gravel-dominated substrates and specific depth ranges, presumably associated with spawning activity. During summer, Shortnose Sturgeon were more dispersed, associating with muddy habitats, whereas in fall/winter, sturgeon congregated in specific regions of the river. These data demonstrate that river use and habitat associations vary seasonally and identify important areas for managing overlap between seasonal habitat use by Shortnose Sturgeon and human activity on the river.