Tree shrews are small mammals living in the tropical forest of Southeast Asia. The habitus of species within the genus Tupaia is often quite similar, so that it is difficult to differentiate the species based on their morphology. We applied comparative bioacoustics, a tool successfully used to discriminate cryptic species of nocturnal mammals, to investigate whether species in the diurnal genus Tupaia can be recognized noninvasively on the basis of a conspicuous loud call, the chatter. We studied to what extent the chatter call of 2 tree shrew species, Tupaia glis and T. belangeri, differed in acoustic structure. We also acoustically analyzed the chatter call of T. chinensis, a subspecies or closely related parapatric species of T. belangeri. Analyzed acoustic features allowed assigning chatter calls with a probability of more than 73% to the species that produced them. Bioacoustical differences are in line with subtle morphological differences, supporting species status for all 3 studied tree shrew species and corroborating immunodiffusion and genetic data that differentiate T. glis and T. belangeri. Loud calls may offer a reliable noninvasive tool for species diagnosis and discrimination in cryptic species of this diurnal mammalian group.