Background: in recent years, market attention has increasingly focused on improving the quality of dairy products. Therefore, animal selection for healthier milk fatty acid composition are therefore of interest. Objective: the aim of this study was to identify whether two polymorphisms (c.878T>C in the SCD gene and g.17924A>G in the FASN gene) are related with the fatty acid composition of milk. Methods: individual milk samples were collected from 50 Chilean Black Friesian cows. Fatty acids (FA) from each milk sample were extracted, analyzed, and quantified (mg FA/g of milk) by gas chromatography. The genotype of each sample was also determined using PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism). Results: milk from cows with a c.878CC genotype in the SCD gene exhibited a greater content of C14:1, C17:1, C18:1n9t, C18:1n9c, and total monounsaturated fatty acids (MUFA), as well as a higher 14:1/14 ratio than other SCD genotypes. In addition, considering the importance of the predominant isomer of conjugated linoleic acid for human diet, we identified a higher content of cis-9, trans-11 conjugated linoleic acid in milk samples from animals that had the c.878CC genotype. For the FASN gene, cows with the g.17924GG genotype presented high contents of C14:0, C16:0, C18:0, C18:1n9t, C18:1n9c, and MUFA. Conclusion: therefore, these polymorphisms could be useful genetic markers for studying the fatty acid composition of dairy milk.