A newly-developed molecular method, hierarchical oligonucleotide primer extension (HOPE), was used to analyze various groups within the species Cylindrospermopsis raciborskii. PCR-amplified internally transcribed spacer sequences of 16S-23S from C. raciborskii in reservoir samples of Taiwan and Kinmen were examined. One of eight sequevars in the clone libraries was closely related to strains obtained from the European continent, while the others, designated of Taiwan (TW) type, belonged to a novel group. Optimized HOPE analyses showed that C. raciborskii distributed in different reservoirs with a relative abundance of 0.5% to 76.4% in the cyanobacterial communities. They further detected the concurrence of three C. raciborskii subpopulations, in which European and TW groups were predominant. The TW sequevars accounted for greater than 87.5% of C. raciborskii in the reservoirs Taihu, Yangmin, Jinsha, and Mudan, while this decreased to 55.4-58.1%, accompanied by a proportional increase of the European group, in reservoirs Lantan and Renyi. These findings revealed the complex subspecies structure within C. raciborskii and the subspecies dynamics associated with geographic locations.