Vocalization is an important feature in anuran identification that could vary among individuals and populations. We present an investigation of multiple-level variation on advertisement calls of Microhyla fissipes from nine populations across Hainan Island and further test the differences between the two geographical groups that were divided by morphological features in a previous study. We found that dominant frequency is the most static call parameter at the individual-level. Four of six call parameters show significant differences between groups and could be useful for identification among groups. The southwest (SW) group from Hainan Island represents the highest dominant frequency among all reported advertisement calls in the literature implying a need for re-evaluation of its taxonomy position.