Taxonomic determination based on morphology alone has failed to describe the evolutionary history of Loxoblemmus appendicularis Shiraki complex in Taiwan. Phylogenetic analysis using the 16S rDNA sequence reveals that three evolutionary lineages of L. appendicularis have been found to coincide with their area of geographical distribution: the Southern, Eastern, and Northern populations. Sequence distance was equal between the Northern and Southern and Northern and Eastern populations (0.032), whereas between the Southern and Eastern populations, the sequence distance was 0.026. Cross-breeding among these three populations has produced abnormal hybrids, suggesting that a possible postzygotic isolating mechanism exists. Biogeographical history suggests the speciation event in L. appendicularis began in the early Pleistocene (1.8 million yr ago [Mya]). Vicariant event created by the rise of the Central Mountain Range >1 Mya led to two separate Eastern and Southern lineages. The following glacial event and formation of a land bridge between Taiwan and the Chinese continent at the end of Pleistocene reintroduced L. appendicularis, currently known as the Northern population, to western and northern Taiwan. Results of sequence divergence, phylogenetic inferences, geographical distribution, and cross-breeding strongly show a current taxonomic recognition of a single species with three parapatric cryptic species.