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Acanthophora spicifera (Vahl) Børgesen is the most widespread and invasive alien macroalga on coral reefs throughout the main Hawaiian Islands. This alga disperses from harbors and ports to coral reefs throughout the state, producing high quantities of biomass that affect a wide range of reef flora and fauna. Population samples of A. spicifera from across the main Hawaiian Islands were collected and compared through two kinds of analyses: DNA sequencing ( based on a variable region of the nuclear large subunit ribosomal RNA gene, and the mitochondrial cox 2-3 spacer region) and fragment techniques (Inter- Simple Sequence Repeats [ISSRs]). DNA sequencing revealed no variation for the two markers, even when collections from other areas of the Pacific and Australia were included. In contrast, ISSR analyses revealed highly structured Hawaiian populations of A. spicifera with a substantial range of both within- and among-population variation, with individual plants forming discrete clusters corresponding to geographic locality.