These data were compiled to describe population reproductive structure of rainbow trout in the Colorado River downstream of Glen Canyon Dam, Arizona. Objective(s) of our study were to describe the population on reproductive structure of the rainbow trout population in Glen Canyon on the Colorado River and evaluate the accuracy of nonlethal methods (manual expression and ultrasonography) for assigning sex and reproductive phase. These data represent samples collected in 3 sampling trips fall in October to November 2018, February 2019, and April 2019. These data were collected in Glen Canyon, from Glen Canyon Dam to Lees Ferry on the Colorado River. Specifically, we sampled three reaches within Glen Canyon to represent the upper (A: 2.0 - 4.8 km downstream of the dam), middle (B: 8.7 – 11.2 km), and lower (C: 18 – 20.7 km) sections of the tailwater, representing a little more than 1/3rd of the 25-km length of Glen Canyon. These data were collected by the study authors (Crossman, Webb, and Korman) as part of ongoing USGS Trout Recruitment and Growth Dynamics sampling trips in 2018 and 2019. Rainbow trout were sampled from each sampling site on each trip and approximately equal numbers taken from each of four size classes (100-199, 200-299, 300-399, 400-499 mm fork length). Fish were measured for length and weighed and a piece of gonad tissue was preserved for histological assignment in the lab. These data can be used to describe population reproductive structure of rainbow trout, understand less invasive methods for assigning sex, and improve our understanding of how somatic growth and energetic status influence population reproductive structure, reproductive seasonality, and later recruitments.