Several lipogenic genes have been shown to have effects on lipid metabolism: stearoyl CoA desaturase 1 (SCD1) catalyzes the desaturation of several fatty acids (FA) in the cis-Delta(9) position in mammary glands of ruminant animals, diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol synthesis in the mammary gland, and sterol regulatory element binding protein (SREBP-1) is a transcription factor that regulates expression levels of the SCD1 gene and other genes relevant to lipid and FA metabolism in adipose tissue and mammary gland. In this work, 351 Italian Brown cows were genotyped for polymorphisms in the SCD1, SREBP-1, and DGAT1 genes to reveal the allelic distribution in the population. Subsequently, effects on individual milk FA composition and on cis-9 unsaturated/saturated FA ratios, a proxy of mammary stearoyl CoA desaturase activity, were investigated. The genotypes of SCD1 (A293V) and DGAT1 (K232A) were determined by an approach based on the ligation detection reaction and a universal array, whereas the genotype of SREBP-1 (84-bp insertion-deletion) was revealed by PCR amplification of intron 5. The genotype analysis showed an unbalanced distribution of alleles within all genes, being the allele with higher gene frequency at 82, 84, and 98% for SCD1, SREBP-1, and DGAT1, respectively. Significant associations between SCD1 and DGAT1 polymorphisms and milk FA composition were found, whereas SREBP-1 polymorphism was not associated with milk FA composition. In particular, SCD1 showed significant association with C14:1 cis-9 and C14:1 cis-9/C14:0, which is considered the best proxy of the desaturation activity in mammary gland. The DGAT1 polymorphism had the strongest association with milk FA composition, which confirmed the key role of DGAT1 in lipid metabolism of mammary gland. However, the unbalanced distribution of alleles in all polymorphisms investigated suggested that the size of population should be increased to confirm the results of the present study.