The population genetic variability of brackish water species is of fundamental importance for the understanding of their evolutionary history and ecological adaptation to drastic environmental changes. Gracilaria tenuistipitata is a euryhaline and eurythermal species that is used in seaweed salads and agar production in Southeast Asia. However, the genetic variation of this brackish water species remains understudied. Here, we investigated the genetic variability and phylogeography of G. tenuistipitata in Southeast Asia using mitochondrial 5′ region of cytochrome c oxidase subunit I sequences. A total of 16 haplotypes were obtained from 161 specimens including 90 newly sequenced from Vietnam. Haplotype and nucleotide diversity of G. tenuistipitata were similar to those of congeneric marine species. Haplotype network analyses revealed a star-like structure including three haplotypes shared by two to four countries, indicating population connectivity as well as population expansion. At the species level, both mismatch analyses and Bayesian skyline plots revealed a demographic or range expansion in the middle Pleistocene rather than demographic bottlenecks. Migration analysis revealed that G. tenuistipitata populations dispersed southwards and northwards from Vietnam. The combination of genetic variability and phylogeographic analyses revealed that the current populations of G. tenuistipitata have been shaped by a combination of climate, sea-level change, and seasonal monsoon currents.