Two species of the calanoid copepod genus Bestiolina occurred in estuarine/coastal waters of the Nansei Islands, southernmost Japan. One of them is described herein as Bestiolina okinawae sp. nov., which is distinguished from known congeners by a combination of the following characteristics: 1) fifth pedigerous somite bearing groups of tiny spinules on the posterior end, 2) female mandible bearing a rod-shaped first gnathal tooth, 3) male left leg 5 with short apical spine on long terminal segment, and 4) legs 23 with endopodal segment 2 lacking spinules. The new species was the dominant zooplankter in oligohaline waters. The other species is morphologically identifiable to Bestiolina similis (Sewell, 1914). Genetic analysis using COI revealed that the two species differed by 9.910.0%, in contrast to small intra-specific genetic variation (0.01.5%). The two species were spatially segregated in low-salinity (230) estuaries and high-salinity (2536) coastal waters, respectively, but co-occurred in some samples. Their body length decreased from the winter (around 20°C) to the summer (around 30°C) by 9.518.1%, depending on the species and sex. In both species, adult males, which lack mandibular gnathobase, were larger than the adult females.