Assessments of novel capture techniques are important to wildlife research. We used Comstock traps, a new live-capture technique, to capture North American river otters Lontra canadensis. We measured Comstock trap functionality in terms of river otter capture efficiency, furbearer capture efficiency, nonfurbearer capture efficiency, and malfunction rate. During 2014–2016, we captured 36 river otters (19 male, 17 female) in Comstock traps during 2,533 trap nights (1 capture/63 trap nights) at Crab Orchard National Wildlife Refuge in southern Illinois, USA. Eleven of 20 (55%) river otters assessed for capture-related injuries received an injury as a result of capture in a Comstock trap. The most common injury was claw loss (45%), followed by tooth fracture (25%) and lacerations (10%). The ease of setting Comstock traps and of releasing nontarget captures made them an appealing option for river otter live capture; however, two river otters died because of hypothermia, two died because of drowning, and one died because of traumatic injuries sustained during capture. Special care should be taken when selecting locations to set Comstock traps with regard to temperature and fluctuating water levels. Researchers attempting to live-capture river otters using this method would benefit by restricting their use to locations with predictable water levels and seasons with mild weather patterns.