Amphisamytha has five currently recognized species. One of these, A. galapagensis has been reported from numerous hydrothermal vents and methane seeps across the Pacific Ocean. Here, a collection of Amphisamytha from a range of Pacific habitats, as well as Amathys lutzi from Atlantic hydrothermal vents, were studied using morphology and DNA sequences from mitochondrial (COI and 16S rRNA) and nuclear (18S rRNA) genes. Phylogenetic analyses revealed a deep-sea clade comprised of the previously recognized Amphisamytha species, and three lineages that are treated as new species. The morphologically distinct Amathys lutzi was nested within Amphisamytha and the monotypic Amathys is treated as a junior synonym of Amphisamytha, with a resulting name change to Amphisamytha lutzi comb. nov. A revision of the other Amphisamytha species, the description of three new species, and a key are provided. The geographic range of the cosmopolitan' A. galapagensis is restricted to hydrothermal vents of the East Pacific Rise and the Galapagos Ridge. Amphisamytha fauchaldi, previously only known from sedimented hydrothermal vents of the Guaymas Basin (Gulf of California, Mexico), is recorded from cold seeps off the coasts of Costa Rica and Oregon. Amphisamytha carldarei sp. nov. is described from hydrothermal vents of the northeast Pacific (Juan de Fuca), while A. jacksoni sp. nov. spans much of the East Pacific Rise and is sympatric with A. galapagensis for part of its range. Amphisamytha julianeae sp. nov. is found at western Pacific vent systems in sympatry with A. vanuatuensis. Relaxed molecular clock analyses were calibrated using the vicariant event involving the Farallon-Pacific Ridge in the northern Pacific Ocean. This provides mean dates for the origin of the deep-sea Amphisamytha clade at 44 or 55 million years ago, and the separation of the PacificAtlantic sister-species pair at 13 or 21 million years ago, depending on the tree used.