During breeding season in May, 2012, we recorded and analyzed the courtship calls and the acoustic parameters of the male Microhyla ornate in Lishui, Zhejiang province using an IC recorder (SX950) and the sound software Praat. We observed that the male M. ornate produced calls characterized by a single harmonic call structure, multiple pulses (7, 9?16) and spindle amplitudes. Analyses of the calls revealed several interesting findings: the dominant frequency of all calls ranged from 1.22?4.09 kHz (n=233); their average values composed of different numbers of pulses were similar and that call duration increased with numbers of pulses; and pulse duration among different multi-pulse call groups was nearly identical, but it was equal or less in the last pulse than other pulses. By contrast, our analyses also showed that pulse interval was negatively correlated with call duration, i.e. the shorter in call duration, the longer in pulse interval among the different numbers of pulses call groups. Within the 7-pulse call group, calls exhibited the largest in pulse interval and the smallest in pulse rate, while those calls in 16-pulse were the opposite. Except that the 7- and 16-pulse call groups were both significantly different in pulse rate with other multi-pulse call groups, we found that pulse rates expressed different variations between pair-wise comparisons of the remaining call groups. As for call intensity, significant differences were only found between the 16-pulse call group and the other call groups, while pairwise comparisons of the remaining groups were not significant. After conducting our initial analyses, we compared the calls of the M. ornate population from Lishui with populations from five other localities—Hangzhou, Xuancheng, Karnoor, Bajipe and Padil—and found that the call structures were similar among the different populations, being single harmonic, while we observed differences in call dominant frequency, call duration, pulse duration and pulse rate among the six populations. This research should prove useful to in furthering our understanding of the acoustic properties of species-specific calls as well as their behavior and the evolution of their communication, thereby providing a robust insight into the reproductive traits among different geographical populations of a species.