In this study we investigate hemipenial variation through ontogeny by preparing specimens of known-aged individuals from captive-bred Plains Gartersnakes, Thamnophis radix, descended from a wild population in northern Illinois, USA. We examined males at two different ages (215–254 days, N = 9) and (829–867 days, N = 12) to compare both juvenile and adult morphologies. Hemipenis length increased isometrically with tail length, and there were no significant differences detected between right and left hemipenis length or width. In addition, this study is the first to explore variation in hemipenial morphology within and among litters. We found significant litter effects on hemipenis length, on the elevation (but not the slope) of the relationship between hemipenis length and tail length, and on number of basal hooks, suggesting a possible genetic basis to these characteristics. These results highlight the importance of examining multiple males through ontogeny, as well as reporting body-size measurements for all specimens, to obtain an accurate representation of the hemipenis morphology of a species for comparative ecological, taxonomic, and evolutionary studies.