Sažetak This study was conducted to describe the single nucleotide polymorphism (SNPs) within exon 8 and its intron 9 flanking region within bovine Diacylglycerol Acyltransferase 2 (DGAT2) gene and the possible association of these SNPs with the milk productive traits. Blood samples were collected from randomly selected Holstein cattle. Genomic DNA was extracted, and a pair of specific PCR primers was designed to amplify a segment that consists of partial exon 8/partial intron 9 of DGAT2 gene. SSCP experiments were optimized and performed for each amplified PCR fragment. Each set of SSCP resolved bands was sequenced and analyzed. Four SSCP patterns representing four genotypes (BD, BB, CD, and AD) were detected with four alleles. Several novel genetic polymorphisms were discovered. Three SNPs (157 C/A, 158 T/G, and 159 G/A) were found in genotype BD, while one SNP (94 G/T, 153 C/A, 154 T/A) were found in the genotypes BB, CD, and AD, respectively. The only non-synonymous SNP was found in genotype CD (344 D/Y), and the effect of this missense mutation on the protein three-dimensional structure was visualized. The sequence homology between the bovine DGAT2 gene and other species was also analyzed. The significance of the correlation of each genotype with the productive traits of milk was observed. The Holstein cattle with BD genotype produced significantly more milk for all studied 90 days of lactation, with a significant effect on fat for only the last 30 days of lactation, whereas there was no effect on protein and lactose percentage for the entire lactation period. The results suggest a novel association between bovine DGAT2 genetic variability and the milk yield in Holstein cattle. This opens interesting prospects for future DGAT2 based selection programs and preservation strategies.