Studies of vocal signals can offer useful information for avian systematics and biogeography. Geographic variation in songs within species has the potential to induce assortative mating, promote reproductive isolation, and consequently leads to divergence between populations and even speciation. Song playback experiments can be of great use in the studies of inferring phylogenetic relationships and assessing taxonomic ranks in birds. Here we carried out song playback experiments in a Wuhan population of great tit (Parus major minor, belonging to the minor subspecies group which is mainly from China) to explore whether the subjects can distinguish songs of its own subspecies group from songs of a foreign subspecies group, the major subspecies group, which is from the west and the north part of Eurasia. By means of comparisons of the subjects’ responses toward the song playbacks of the major and minor subspecies groups, we explored whether a potential behavioral isolation exists between them. We tested 24 male great tit individuals from nonadjacent territories in Wuhan, China, from March to May, 2014, with 12 birds tested for song playbacks from each group. Songs used for playbacks were of different song types and from different locations in the distribution ranges of both subspecies groups (see Fig. 1, Appendix 1). Each of the 24 song playback files was only for a single use as stimulus in a great tit territory. We used both qualitative and quantitative behavioural parameters to assess the responding behaviour of our subjects during and after the playback. We used Fisher’s Exact Test for comparisons on qualitative parameters between groups. Mann-Whitney U tests were used for comparisons on quantitative responding behaviour parameters between groups. The results suggest that the difference in responses to the playbacks of the two subspecies groups is significant (see Fig. 2, Table 1). The responses to the minor song playbacks are generally intense while the responses to the major playbacks are weak and even there’s no response at all in most subjects (see Fig. 2, Table 2). Our results indicate that a considerable acoustic divergence, which can act as a potential reproductive isolation mechanism, exists between the major and minor subspecies groups of great tits, and thus support the suggestion of separate species in the Parus major complex.