Reintroduction projects often expose animals to a series of acute stressors that may cause chronic stress and lead to the stress response. The stress response results in the release of glucocorticoids that, when excessive, can cause detrimental effects to the animal. Glucocorticoids can be extracted from feces and quantified as an effective method for assessing stress levels. We collected scats from 10 river otters (Lontra canadensis; 3 from MD and 7 from NY, USA) held captive for the Pennsylvania River Otter Reintroduction Project (PRORP). We used these scats to verify the use of the Correlate-EIA^TM Corticosterone Enzyme Immunoassay Kit (Assay Designs, Inc., Ann Arbor, MI) to evaluate stress levels in otters. We also determined trends in stress levels during the initial 10–12 days otters were in PRORP captivity, and compared glucocorticoid levels for 5 of the New York otters the morning before, the morning of, and the morning after veterinary examinations to determine if associated procedures (e.g., physical and chemical restraint) caused increased stress levels. Glucocorticoid concentrations declined from time 1 to time 2 for the 3 otters from Maryland (an average decline of about 6-fold) and for 5 of 7 otters from New York. Among otters evaluated for stress associated with veterinary examinations, average glucocorticoid concentrations were increased the morning of and the morning after veterinary examinations from the day before the veterinary examinations. We demonstrated that fecal glucocorticoids are an effective method for assessing stress levels in otters and that PRORP's captive management program did not contribute to increasing stress during the 10–12-day evaluation period. Fecal glucocorticoid assays could be used to evaluate stress levels of zoo or permanently captive otters, determine the most effective husbandry techniques for housing otters, and evaluate effects of both management practices and environmental conditions in the wild and in captivity.