Due to the sparse and unstable nature of insular freshwater habitats, marine larval dispersal of amphidromous species is considered a critical element of population persistence. We assessed population genetic structure of freshwater prawn Macrobrachium lar across its range that encompasses two biogeographic barriers: the vast open ocean separating Western and Central Pacific regions and the Indo-Malay archipelago separating Indian and Pacific oceans. A total of 173 samples collected from 21 islands throughout the Indo-Pacific were sequenced at 16S and 28S rDNA. We observed distinct genetic isolation of populations located at the eastern and southwestern edge of the species range but no evidence of an effect of the Indo-Pacific barrier. Differentiation patterns are consistent with a stepping-stone model of dispersal. Genetic differences of Central Pacific populations may reflect founder events associated with colonization of isolated islands, or be a signature of a past bottleneck after population depletion caused by drastic climatic events.