The Marajoara breed of horses is an important animal group genetically conserved on the island of Marajó, State of Pará, Brazil. This breed exhibits very specific characteristics, but has suffered decharacterization as a result of crossing with other naturalized breeds in Brazil. For this reason, Marajoara horses have been introduced into the National Genetic Conservation Program, which includes both ex situ and in situ preservation, and also involves establishing productive, reproductive, genetic and phenotypic parameters for Marajoara horses. Therefore, the aim of this study was the cytogenetic characterization of the Marajoara horses. In order to do it we used the peripheral blood lymphocyte cultures of eight specimens, evaluated in terms of diploid number, morphology, chromosome biometry and C-and NOR-banding patterns. All specimens evaluated had 2n=64 chromosomes and karyotype consisting of submetacentric pairs (1 to 11, 13 and X), a metacentric pair (12) and telocentric pairs (14 to 31 and Y). Cbanding was observed marking centromeric of all complement and interstitial marking feature for X and Y chromosome almost completely heterochromatic. Nucleolus organizer regions were observed in pair 1 in the telomeric region, 28 in interstitial short arm and in 31 near the centromere. The cytogenetic characterization of these animals is the first step in the process of selecting animals for reproduction by establishing the diploid number as a basis for subsequent actions in the genetic conservation program.