Genetic polyandry was assessed in 27 litters of Blacknose Shark (Carcharhinus acronotus) from genetically distinct populations in the U.S. Atlantic (19 litters) and eastern Gulf of Mexico (eight litters) using 23 polymorphic microsatellite loci. Two methods were used to estimate genetic polyandry, and the overall observed rates were high (74% COLONY2; 81% allele counting method), with a maximum of four sires detected in a single litter. When separated by region, the rate of genetic polyandry was 63% (COLONY2) or 74% (allele counting method) in the U.S. Atlantic, and 100% (both methods) in the eastern Gulf of Mexico. Data were resampled to evaluate how the number and diversity of markers analyzed affected the estimated rate of genetic polyandry. The number of alleles per locus had a dramatic effect on the detection of polyandry, with a difference of 56% in the estimated rate of genetic polyandry (22% vs. 78%), when using only the five loci with the highest diversity (A = 21–44) versus lowest diversity (A = 3–5). The total number of markers assessed also affected the estimated rate of genetic polyandry, and the estimated rate increased by 13% when using 10 versus 20 loci (66% vs. 79%, respectively). These results suggest that unless loci are highly polymorphic, relatively large numbers of loci are required to estimate the rate of genetic polyandry accurately in elasmobranchs, particularly those with small litter sizes like the Blacknose Shark.