The effect of polymorphism of genes involved in the milk fatty acid synthesis, Stearoyl CoA Desaturase 1 (SCD1), 1-Acylglycerol-3-phosphate-O-acyltransferase-6 (AGPAT6), Fatty Acid Synthase (FASN), and Leptin (LEP) on fatty acid (FA) and chemical composition of milk in Podolian and Italian Holstein bovine breeds was investigated. Principal results in Podolian cattle breed highlighted for the VV genotype of SCD1 the highest protein and casein content. The TT genotype of LEP gene, AW/AR diplotype for FASN gene, and TT genotype for AGPAT6 gene revealed the lowest fat content. For SCD1 gene the lowest medium chain FA (MCFA), and Thrombogenic Index (TI) were found in the AA genotype. The LEP TT genotype showed lower saturated FA (SFA), n6/n3, Atherogenic Index (AI), and SCFA, and higher polyunsaturated FA (PUFA), long chain FA (LCFA), n3, n3/n6. Furthermore, the CT genotype evidenced lower PUFA, MCFA, LCFA, n3, and higher CLA. The SCFA were higher, and MCFA were lower in the AW/AR diplotype of the FASN gene, and SCFA were the lowest in the AR/AR. The AGPAT6 CT genotype showed the lowest value of PUFA, and n3, and the highest of n6/n3; CC genotype showed the highest content of PUFA, and n3. Podolian milk revealed higher content of PUFA, n3, CLA, PUFA/SFA, and lower n6/n3, TI, and SCFA than milk from Italian Holstein, evidencing a better nutritional value for human health.