Studies determining and categorizing ultrasonic calls in mammals have generally been limited to laboratory experiments with mice and rats or field research on bats. However, recent studies have discovered high-frequency sounds in several mammalian taxa. We describe the vocal repertoire of high-frequency calls in two species of North American flying squirrels (Glaucomys sabrinus and G. volans). We collected passive recordings from captive colonies of G. sabrinus and G. volans to generate a library of calls for each species. Using visual and auditory assessments, as well as quantitative measurements (i.e., spectral and temporal characteristics), we identified 10 specific call-types for G. sabrinus and 27 specific call-types for G. volans. The most common call-types emitted by G. sabrinus included arc chirps, tonal chirps, two-toned chirps, trills, and upsweeps; and for G. volans included arc chirps, barks, descending crows, tonal chirps, and trills. Additionally, we recorded two call-types made by G. volans juveniles: trills and tonal chirps. Most call-types emitted by G. sabrinus were also emitted by G. volans, showing overlap in the repertoire of both species, although variation between species is evident in certain call-types. We also found both species displayed evidence of frequency alteration (i.e., modification of the frequency of one squirrel's call when simultaneously calling with another squirrel using similar call-types). Our call library demonstrates a complex vocal range of tonal and frequency-modulated calls emitted by both species. Acoustic detection of North American flying squirrels is a new monitoring technique that can be used for conservation and management purposes. Differences in the trills, tonal chirps, and arc chirps provide a means to distinguish calls between these species. Increasing understanding of behavior associated with vocalizations may provide a more complete understanding of flying squirrel ecology, including intra- and interspecific interactions.