There is a dearth of information regarding the vocal repertoire of North American river otters (Lontra canadensis). This indicator species is cosmopolitan yet elusive, making recordings methodologically difficult in the wild. Therefore, this exploratory study uses video and audio recordings of two populations of North American river otters in human care to broaden the known vocal repertoire of river otters in various social contexts. The populations consist of a male-female and a male-male pair. This study is the first to examine the vocalizations produced in a male-male pair of river otters. Call types were acoustically distinguished based on their appearance on a spectrogram. Parameters including average duration, frequency (high, low, max, 1st quarter, center, and 3rd quarter), and power (max and average) were measured for each call. Because vocalizations are the focal point of this study, only behaviors co-occurring with vocalizations were included in the chi square analysis that showed a significant relationship between call type and behavior. Squeaks and whines were present during agonistic behaviors while chirps were produced during non-agonistic behaviors including investigating, stationary, and grooming. Results support that behavior likely plays a role in the type of calls produced by river otters in human care.