We collected tissue samples from 41 nesting hawksbill turtles (Eretmochelys imbricata) at Sandy Point National Wildlife Refuge, St. Croix, US Virgin Islands, to characterize the genetic structure of this rookery in terms of mitochondrial DNA; we compared haplotype frequencies from this rookery to those from Buck Island, another hawksbill nesting beach on St. Croix. Pairwise F_ST comparisons showed that Sandy Point was demographically distinct from Buck Island (F_ST = 0.501, p < 0.001), a finding reinforced by significantly different haplotype frequencies (χ² = 51.76, p < 0.001) and a lack of interchange of nesting females between both sites based on mark–recapture data. Our results support the delineation of the nesting populations at Sandy Point and Buck Island into separate units for the purposes of management.