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Knowledge of genetic diversity and relationships between breeds is very important for conservation programs. Hair samples were collected from 393 individual Nordestino horses and genotyped using 14 microsatellite markers in order to investigate the genetic relationship between this breed and 66 international horse breeds. There was high allelic diversity and inbreeding coefficient within population values were not significant, which was probably due to crossbreeding. Despite the Nordestino horse population being in Hardy-Weinberg equilibrium, a global deficit of heterozygotes was observed. This may represent evidence of repeated use of the same stallions for breeding, which is consistent with the high number of castrated males found. Campolina, Mangalarga Marchador, and Mangalarga were the Brazilian horse breeds most closely related to the Nordestino horse, which is a reflection of recent introgressions. Among Iberian horse breeds, the Sorraia breed appears to have had an important influence on the genetics of the Nordestino horse. Those results provide important information that can guide future conservation programs.