Genetic variability and relationships among five cattle breeds (Holstein, Pinzgau, Limousin, Slovak Spotted and Charolais) bred in the Slovak Republic were investigated separately using 11 microsatellite markers and 61 blood group systems. Allele frequency, heterozygosity (Ho, HE) and PIC values were investigated. F-statistics were computed separately. For microsatellite markers FIS, FIT, FST and for blood groups HS, HT, GST parameters were calculated. Microsatellite and blood group comparison showed similar results by F-statistics but some differences were marked using the other methods. Both methods were able to detect close relation between Slovak Pinzgau and Slovak Spotted cattle breeds. Their relation was confirmed by genetic distance, principal component analysis (PCA) and coefficient of admixture (mY). Important divergences between different markers used in the study were observed by the characterisation of Limousin and Charolais breeds.