Alentejano and Iberian breeds are biogeographically located in the southwest of the Iberian Peninsula. These breeds shared a set of common characteristics. Both are phenotypically very similar showing a low growth rate and a high intramuscular fat content and are reared under extensive systems in open-range fields. The aim of the current study, carried out within the framework of the TREASURE project, intends to characterize of the genetic diversity of Alentejano and Iberian breeds using the genotyping data of 32 polymorphisms located on 26 major genes. These polymorphisms were genotyped in a total of 950 animals belonging to Alentejano, Iberian and 18 additional European breeds. The low mean values of observed (Ho) and expected (Hs) heterozygosity and FIS point out a low genetic diversity in the analyzed breeds. The genetic distances estimated using Ds and FST revealed values were close to 0 (0.005 and 0.030, respectively), suggesting that these breeds are genetically similar. The population structure analyzed with multivariate methods such as Discriminant Analysis of Principal Component and admixture analyses showed that both breeds were grouped within the same cluster. These results are supported by other authors based on mitochondrial sequences who reported short genetic distances between these breeds. Although complementary analyses using a larger number of markers should be performed, the results of the current analyses support the hypothesis of Alentejano and Iberian could be different strains of the same breed.