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Computer-assisted semen analysis (CASA) and cluster analysis were used to compare spermatozoa swimming kinematics and milt quality between wild and captive shortnose sturgeon (Acipenser brevirostrum). Milt samples from 27 shortnose sturgeon were collected in May 2016 and June 2017. Of these, 19 were wild caught in the Saint John River, New Brunswick, Canada, and eight were from a captive population at the Mactaquac Biodiversity facility. The following kinematic variables were measured immediately following sperm activation ((-)5s), at 30, 60, and 180 s post-activation; average path velocity (VAP); straight-line velocity (VSL); curvilinear velocity (VCL); amplitude of lateral head displacement (ALH); beat cross frequency (BCF); straightness (STR); linearity (LIN); wobble (WOB); percent motility (MOT). Analyses were conducted at 7, 10, and 14 degrees C to determine potential effects of temperature on kinematics. Principal components analysis (PCA) of original kinematic variables yielded two main components, a speed/wobble component along with a movement pattern component. Hierarchical cluster analysis (HCPC) indicated there were distinct subpopulations, with composition of clusters the result of fish source (wild-caught or captive). Wild-caught fish had greater sperm densities (P = 0.0064) and sperm swimming speeds compared to captive fish (P < 0.05). Temperature had a significant effect only on captive spermatozoa, and this result was not consistent between time periods. There was no effect of hormonal manipulation on spermatozoa motility kinematics. Results indicate there are significant differences in measures of milt quality between wild and captive shortnose sturgeon, indicating an effect of rearing condition on reproductive potential, which may affect fertilization success.