One of the most threatened ecosystems on many islands may be anchialine habitats, or coastal land-locked water bodies with no surface connection to the sea yet containing brackish water that fluctuates with the tides. To better manage these habitats, it is important to develop a broader understanding of the biodiversity within them since such knowledge plays critical roles in establishing conservation strategies. In this study, the genetic variation and population structure of an anchialine atyid shrimp, Caridina rubella Fujino and Shokita, 1975 was investigated in the Southern Ryukyu Archipelago, Japan. Given a planktotrophic larval stage and its potential amphidromous life cycle, populations of C. rubella on the island of Miyako-jima (to which it is apparently restricted) were hypothesized to have little to no structure across the island. To test this, 61 individuals were collected from four anchialine caves and sequence variation examined at the cytochrome c oxidase subunit I (COI) region of the mitochondrial DNA (mtDNA). Surprisingly, significant genetic structure was exhibited across distances ranging from <20 m to > 10 km. Additionally, deep (∼17% p-distance) genetic divergence correlating with distinct variation in rostrum lengths was found between closely situated, but more-or-less completely isolated, populations. This implies “C. rubella” may actually represent two distinct species on Miyako-jima. Given that this atyid is already listed as a threatened species by the Japanese government, the results presented here are useful in the formulation and implementation of future conservation plans for populations of C. rubella on Miyako-jima.