Species that overlap in their morphologies are sometimes difficult to distinguish from one another, which can complicate species’ conservation and management. The Little Brown Bat (Myotis lucifugus) and Yuma Myotis (Myotis yumanensis) are sympatric in parts of their range in western North America, and they overlap in morphology, making them difficult to tell apart in the hand in some areas, such as the Pacific Northwest. We compared various methods of distinguishing between M. lucifugus and M. yumanensis to genetic results, using a blind test approach to remove observer bias. Using multiple independent observers, we used external morphology and echolocation-call structure to classify bats from a maternity colony consisting of both species. Genetic analysis confirmed 13 M. lucifugus and 40 M. yumanensis. Minimum echolocation-call frequency separated 100% of M. lucifugus from M. yumanensis using a cut-off of 43 kHz. All M. lucifugus had a minimum echolocation-call frequency ≤42.8 kHz, whereas M. yumanensis had a minimum frequency ≥44.55 kHz. There was some overlap in forearm length; a cut-off of 36 mm would have correctly identified 77% of M. lucifugus and 100% of M. yumanensis to species. Criteria based on subjective assessment of fur sheen and length as well as ear color were moderately successful (90.5 and 77% success by 2 separate observers) in distinguishing between the 2 species. The use of Munsell soil color charts and multivariate statistics to classify fur and membrane color and confirm species identification was not successful. Our results suggest that mean minimum call frequency alone is sufficient for distinguishing between M. lucifugus and M. yumanensis. Use of quantitative rather than qualitative criteria eliminates observer bias and appears to be better for identifying these 2 species.