The domestic horse (Equus caballus) was re-introduced to the Americas by Spanish explorers. Although horses from other parts of Europe were subsequently introduced, some New World populations maintain characteristics ascribed to their Spanish heritage. The southeastern United States has a history of Spanish invasion and settlement, and this influence on local feral horse populations includes two feral-recaptured breeds: the Florida Cracker and the Marsh Tacky, both of which are classified as Colonial Spanish horses. The feral Banker horses found on islands off the coast of North Carolina, which include, among others, the Shackleford Banks, the Corolla and the Ocracoke, are also Colonial Spanish horses. Herein we analyse 15 microsatellite loci from 532 feral and 2583 domestic horses in order to compare the genetic variation of these five Colonial Spanish Horse populations to 40 modern horse breeds. We find that the Corolla horse has very low heterozygosity and that both the Corolla and Ocracoke populations have a low mean number of alleles. We also find that the Florida Cracker population has a heterozygosity deficit. In addition, we find evidence of similarity of the Shackleford Banks, Marsh Tacky and Florida Cracker populations to New World Iberian horse breeds, while the origins of the other two populations are less clear.