The North American river otter (Lontra canadensis) serves as an indicator species for environmental monitoring, is prized as a valuable furbearer, and is a popular display animal in zoologic collections. Nephrolithiasis has been reported as a frequent problem in other free-ranging and captive otter species but is rarely reported in North American river otters. In this study, we compared the prevalence of nephrolithiasis diagnosed using routine gross pathologic examination techniques with the use of computed tomography (CT) of excised kidneys. We also evaluated whether otter nephroliths could be accurately classified by their CT densities, and we examined the renal tissue uric acid concentrations in free-ranging otters in North Carolina, USA. Kidneys were collected from carcasses of legally trapped, free-ranging animals. Nephroliths were observed in 16.2% of the individuals (n = 229). Associations were found between age and nephrolith status and between capture location and nephrolith status (P = 0.026 and < 0.001, respectively). Computed tomography Hounsfield unit density measurements were not useful in determining nephrolith chemical composition in this study. Renal tissue uric acid concentrations were similar across genders, age groups, and stone status. The chemical composition of the nephroliths was determined by scanning electron microscopy–energy dispersive X-ray spectroscopy to be calcium phosphate in the carbonate form.