Phylogeographic analysis of Scotophilus kuhlii between Hainan Island and Guangdong Province was conducted to validate the existence of two subspecies separated by the Qiongzhou Strait. A total of 37 individuals from 3 assumed populations (8 roosts) were examined using 617 bp mtDNA control region segments. Nineteen individuals from cross-strait populations of Hainan Island and the mainland shared 3 of 13 haplotypes. Overall nucleotides diversity was 0.003, a low genetic variation level compared to previous reports for other chiropteran species. The neutral test, mismatch distribution, and star-like TCS network suggested a bottleneck effect occurring at 21,000 years BP during the last glacial maximum and a subsequent population expansion at 7,000 years BP, corresponding with a global warming period. AMOVA analysis, intertwined haplotypes in the TCS network, and rejection of “Isolation by Distance” by the Mantel test indicated no distinct population structure among these populations. Three populations seem to have been derived from a single panmictic unit and the Qiongzhou Strait did not hinder gene flow. Our result, a striking difference in population structure compared with other sympatric chiropteran phylogeographic reports, may indicate variations in the barrier effect of the strait in different species. Based on these results, we suggest that the cross-strait population should be classified as one subspecies, namely S. k. consobrinus, instead of two traditional subspecies.