The BovineSNP50 genotyping array is a product with a wide range of applications in cattle such as genome-wide association studies, identification of copy number variation and investigation of genetic relationships among cattle breeds. It also holds potential for genomic selection, especially for traits that are expensive and difficult to measure. The successfulness of this chip for any of these applications depends on the degree of polymorphisms in the cattle breeds. The SNP50 array has not been validated in any South African cattle population and this could lead to overestimating the number of polymorphic SNPs available for application in it. This study is a first attempt to evaluate the Bovine64SNP50 genotyping array in the South African cattle population. Ninety six bovine samples, consisting of 45 Holstein, 29 Nguni, 12 Angus and 10 Nguni x Angus crossbred animals, were genotyped with the BovineSNP50 infinium assay. The results of this study demonstrated that 40 555 SNPs were polymorphic (MAF >0.05) in these breeds and indicate potential for application in South African cattle populations. Genomic information generated from the BovineSNP50 can now be applied in genetic prediction, genetic characterization and genome-wide association studies.