•The population does not exhibit serious factors that affect genetic variability.•It is necessary to implement a genetic management program seeking to maximize genetic variability and especially conserve the structure of the Mangalarga Marchador breed as a well-defined genetic entity differentiated from other horse breeds. Mangalarga Marchador (MM) is the most important Brazilian horse breed, with 600 thousand animals registered. The aim of this study carry out the genetic characterization of the breed. Samples were collected from 355 horses MM, n = 218; Andalusian n = 20; Purebred Lusitano n = 21; Sorraia (SOR) n = 16; Thoroughbred n = 21; Campolina (CAM) n = 21, and Mangalarga (MAN) n = 38 and genotyped with the SNP 70K BeadChip. Parameters were calculated, such as expected heterozygosities (He), Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (r2), genetic differentiation, and principal component analysis (PCA); and haplotypes in common and population structure was assessed by Bayesian methods. The MM breed obtained reasonable He and deviations from HWE. The SOR breed had high r2, and CAM and MM had low r2. MAN and MM had a similar haplotype block. Analyses of population structure showed that, just as the other Brazilian breeds, the MM breed is not well defined, showing signs of substructure and possibly admixture. The analyses with PCA showed that Brazilian breeds clustered together, and well separated from the remaining breeds. A breeding program seeking to maximize the variability of MM and structure the breed as a more defined genetic entity should be implemented immediately. Display omitted