The aim of this study was to detect the k232a polymorphism in acyl-Coa:diacylglycerol acyltranferase-1 gene (DGa T1) in the Holstein and Slovak spotted breeds in Slovakia using improved primers and high-throughput microchip electrophoresis (MCe). Samples of 87 animals from the Holstein breed and 42 animals from the Slovak spotted breed were genotyped for DGAT1 K232A polymorphism (a and k alleles) using the PCR-RFLP technique. We observed a clear resolution of three different genotypes following fast, automated analysis of PCR products by microchip electrophoresis. The Holstein breed showed the frequencies 0.8621 for the a allele and 0.1379 for the k allele. Most animals were aa homozygotes (frequency 0.7356) or ak heterozygotes (frequency 0.2529). We found only one kk homozygote (0.0115) in the tested sample. The observed frequency of the allele k in the tested herd of Slovak Holstein is lower than reported for this breed in other countries. The genotyping data regarding k232a polymorphism in the Slovak spotted breed showed even lower frequency of the allele k (0.0476) than in the Holstein breed. The Chi-square test found a deviation from the Hardy-Weinberg equilibrium of the DGAT1 K232A genotypic frequencies for Holstein and Slovak spotted breeds. This disequilibrium could be related either to the sampling of the genotyped animals or can be a result of selection for the milk production. our results could be used to guide further association studies between this locus and milk traits in these breeds.