The polymorphism of six milk protein loci in five Cuban breeds was investigated by means of DNA analysis by combined techniques of PCR and amplification created restriction sites (ACRS), the method of allele-specific oligonucleotides (ASO), and restriction fragment length polymorphism (RFLP). Allele frequencies of loci CASA1, CASAB, CASA2, CASK, LAA and LGB were estimated in the five breeds (N = 324), the number of individuals per population being: Siboney de Cuba (SC = 85), Cuban Creole (CC = 60) Cuban Zebu (ZC = 61), Cuba Mambí (MC = 60) and Taíno of Cuba (TC = 58). Hardy–Weinberg equilibrium was estimated for each locus in each population, showing that all the populations have at least one locus deviated from this condition of equilibrium and always through heterozygote excess. Alleles CASA1C and LAAA were identified in the CC and CT breeds, showing the presence of Bos indicus genes in these populations. An increase of heterozygosis is observed in these populations, and genotype frequencies in each population allow differentiation of these from those which originated them.