This study investigates the fatty acid profiles of milk from three indigenous cattle breeds, Gir, Sahiwal, and Tharparkar, aiming to elucidate breed-specific variations and their implications for nutritional quality. Comparative analysis was conducted using gas chromatography coupled with flame ionization detector (GC-FID). The study revealed significant differences in fatty acid composition among the breeds, attributed to genetic, dietary, and environmental factors. Gir milk exhibited higher levels of medium-chain fatty acids, along with elevated proportions of palmitic acid and monounsaturated fatty acids (MUFAs). Sahiwal milk displayed lower levels of MUFAs, while Tharparkar milk demonstrated the highest content of polyunsaturated fatty acids (PUFAs). These findings underscore the importance of considering breed-specific variations in fatty acid composition for optimizing the nutritional quality of dairy products.