The mitochondrial DNA cytochrome c oxidase subunit I sequences from 95 specimens of Semisulcospira libertina in Taiwan were identified as two major phylogroups, exhibiting a southern and northern distribution, north of Formosa Bank and south of Miaoli Plateau. The genetic distance between these two phylogroups was 12.20 %, and the distances within-phylogroups were 4.97 and 5.56 %. According to a molecular clock of 1.56 % per lineage per million years, the divergence time between these two major phylogroups was estimated at 4.94 million years ago (mya), with the two phylogroups forming at 3.64 and 3.75 mya, respectively. Moreover, the geological events have suggested that Taiwan Island emerged above sea level at 4-5 mya, and became its present shape at 2 mya. These results suggested that these two phylogroups might originate from two independent ancestral populations or divergent before colonizing Taiwan. Within South phylogroup, the initial colonization was hypothesized to be in Kaoping River (WT), followed by its northward. The high divergence between south- and north of WT River was influenced by the formation of the Kaoping foreland basins. Within North phylogroup, the colonization was from central sub-region through paleo-Miaoli Plateau to northern and northeastern sub-regions. This study showed that the landform changes might have shaped the genetic structure of S. libertina in concert. Apparently, two cryptic species or five different genetic stocks of S. libertina could be identified; these results are useful for the evaluation and conservation of S. libertina in Taiwan.