The lowermost dam on the Penobscot River, Maine, was removed in 2013, making new habitat available for migratory fish. There is no evidence that endangered Shortnose Sturgeon Acipenser brevirostrum have spawned in the Penobscot River in recent years, but dam removal has facilitated access to potential freshwater habitat essential for spawning. Spawning success also depends on the quality of the available habitat. We sought to describe the distribution and amount of suitable spawning habitat in the first 5-km reach upstream of the removed dam. Previously collected river elevation and bottom substrate data were used to create two-dimensional hydrodynamic simulations of the reach for spring discharges ranging from 310 to 1480 m3 s-1 using the program River2D. Simulations were validated and adjusted using field-collected data. Suitable spawning habitat was predicted based on literature-informed suitability curves of depth, velocity, and bottom substrate. Between 41% and 63% of the study area offered usable spawning habitat, depending on river discharge. Velocity was the most limiting characteristic to overall suitability at all modeled discharges. Embeddedness was minimal at suitable sites. Based on the habitat characteristics considered, the newly accessible reach of the Penobscot River could support Shortnose Sturgeon spawning, offering critical habitat for this endangered species.