The longbill spearfish (Tetrapturus pfluegeri; Istiophoridae) is an epipelagic billfish species broadly distributed in the Atlantic Ocean and exploited by multinational fisheries. No data exist on the population structure or genetic diversity of this apex predator, despite a strong need for this information to inform international conservation and management efforts. Thirteen microsatellite loci, comprising mostly tri- and tetra-nucleotide repeats, were isolated and characterized for this species by genotyping individuals (n = 29–42) obtained from western North and South Atlantic fisheries. The average number of alleles ranged from 3 to 16 and the expected heterozygosity ranged from 0.35 to 0.86. This suite of markers provides the first population genetic nuclear resources for the longbill spearfish, allowing initiation of studies for assessing the population structure and demography of this poorly understood species.