Echolocation is the use of reflected sound to sense features of the environment. Here, we show that soft-furred tree mice ( Typhlomys) echolocate based on multiple independent lines of evidence. Behavioral experiments show that these mice can locate and avoid obstacles in darkness using hearing and ultrasonic pulses. The proximal portion of their stylohyal bone fuses with the tympanic bone, a form previously only seen in laryngeally echolocating bats. Further, we found convergence of hearing-related genes across the genome and of the echolocation-related gene prestin between soft-furred tree mice and echolocating mammals. Together, our findings suggest that soft-furred tree mice are capable of echolocation, and thus are a new lineage of echolocating mammals.