The aim of this study was to determine the animal CYP19/PvuII genotypes, evaluate the genetic structure of population and estimate the effect of genotypes on long-life milk production traits in Slovak Simmental cattle. Blood samples were obtained from in total 150 Slovak Simmental cows originating from four different farms localized on eastern Slovakia. The polymorphism in CYP19 gene was detected using PCR-RFLP method and PvuII restriction enzyme. In cows population was identified the presence of all three genotypes: AA (74.7%), AB (21.3%), and BB (4%). The A allele was more frequent (0.85±0.02) than B (0.15±0.02). In population was found deviations from HWE. The level of locus polymorphism in the analysed population was only low (PIC=0.212). The activity of CYP19/PvuII alleles were unbalanced (Ne=1.33) and the higher proportion of homozygous animals caused the decrease of observed heterozygosity (He=0.21). Based on F – statistic results the most of the genetic variation was distributed within all analysed individuals (FIT 0.155). Evaluation of CYP19/PvuII genotype effect on milk production traits was carried out with linear models (GLM procedure). Based on the selected effect we were able to estimate the variability of analysed traits on in average 85%. The CYP19/PvuII genotype affected significantly the variability of milk and protein yield (P<0.05). The higher production of both traits was found in individuals with BB genotype. Statistically significant influence was detected also for sire (P<0.05), breeder (P<0.0001) and total number of completed lactations (P<0.0001).