Species delimitation is an important issue in evolutionary biology. Acoustic and morphological traits are vital to frogs, especially in species recognition and reproduction. Japanese tree frog (Buergeria japonica) is widely distributed from Ryukyu Archipelago to Taiwan. Recent studies have showed high genetic differentiation among islands, with non-monophyletic relationship among populations in Taiwan. This indicated the necessity to re-evaluate the taxonomic status of B. japonica across regions. To examine the variation among populations, we sampled 17 localities from Ryukyu and Taiwan, analyzed their acoustic traits of long calls and short calls, and 19 morphological traits. We found that the southwestern and eastern populations in Taiwan represent a unique type of long call which is different from the remainders. Furthermore, duration and rise time of short calls in southwestern and eastern areas are shorter. In morphology, results showed that the principal components head traits from southwestern and eastern group in Taiwan is significantly higher than the others. These results indicated that there is a cryptic species in Taiwan which represents different acoustic signal but with similar morphology from B. japonica sensu stricto.