Twenty homologous microsatellite markers, or simple sequence repeats (SSRs), were developed for blackfin tuna (Thunnus atlanticus) through the use of a direct Seq-to-SSR approach. The number of alleles per locus ranged between 5 and 26, and the expected heterozygosity ranged between 0.640 and 0.969. Three loci displayed significant departure from Hardy-Weinberg equilibrium expectations, likely reflecting occurrence of null alleles. Another locus showed consecutive alleles that differed by one base pair only. Consequently, this locus may be prone to elevated rates of scoring errors. The remaining 16 loci will be valuable for studies in conservation genetics of blackfin tuna.