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Abstract Deep-sea mining of hydrothermal deposits off the coast of Japan is currently under consideration, and environmental baseline studies of the area are required to understand possible impacts. The aim of this study is to clarify population structures of dominant benthic megafaunal species near hydrothermal vent fields in the Okinawa Trough, using a population genetics approach. We examined dominant deep-sea scavenging species including eels, several amphipods, and a decapod and performed population genetic analyses based on the mitochondrial cytochrome c oxidase subunit I region. Several sites were sampled within Okinawa Trough to examine intra-population diversity while two other locations 1400–2400 km away were chosen for inter-population comparisons. For synaphobranchid eels Simenchelys parasitica and Synaphobranchus kaupii, our results showed significant intra-population diversity but no inter-population genetic differentiation, suggesting strong genetic connectivity and/or large population sizes. In addition, single nucleotide polymorphism analysis also confirmed strong genetic connectivity for Simenchelys parasitica. Among scavenging amphipods, we detected seven putative species using molecular phylogenetic analysis. We evaluated population structures of the most abundant species of amphipods and a decapod species (Nematocarcinus lanceopes). Our results provide basic information on the genetic population structures of benthic megafaunal species near hydrothermal vent fields, which can be used to select candidate species for future connectivity analysis with high-resolution genetic markers and aid understanding of the potential population impacts of environmental disturbances.