To understand spatial patterns of genetic differentiation in the Brachionus calyciflorus species complex, the rDNA ITS regions of 124 rotifer clones collected from eight geographic regions in East China were sequenced and analyzed. A total of 73 haplotypes were defined, and were grouped into three clades by the phylogenetic trees. The divergences of ITS sequence among the three clades ranged from 4.2% to 25.3%, indicating the occurrence of three cryptic species (cryptic species I, cryptic species II and cryptic species III). The sympatric co-occurrence of these cryptic species was observed at Guangzhou, Danzhou and Wuhu. The nucleotide diversity in B. calyciflorus complex was higher, and so was haplotype diversity. Within cryptic species III, the higher genetic differentiation might be attributed to fragmentation and restricted gene flow. In addition, the rapid increase of colonizers after an ambiguous glacial bottleneck might interfere with effective gene flow and accelerate genetic differentiation of geographic populations. The nested clade analysis suggested to some extent the geographic structure, and the non-significant correlation existed between the geographic distance and the average pairwise difference between populations (Da) as well as Fst. The Younger Dryas(YD) Event after the last full glacial period might has considerable effect on the patterns of geographic structure and distribution of cryptic B. calyciflorus species. During the YD event, the three cryptic species probably retreated and co-occurred in multiple relict refugia to the south of Nanling area. Long-distance colonization possibly coupled with subsequent fragmentation might be the main reason of the current patterns of geographic structure. The geographic distribution of B. calyciflorus complex on a global scale might be attributed to the motion of continental plates.