Human milk (HM) of healthy, well-nourished, lactating mothers is a unique and ideal source of nutritive factors, like hormones, cytokines, chemokines, growth factors that ensures the proper growth and development of infants. Among the main components of HM, fat is an important energy source and a regulatory factor. The quality of milk fat depends on its fatty acid (FA) composition. Gas chromatography coupled with flame ionisation detection is one of the most common methods for analysis of the FA profile of HM. The aim of this study was to evaluate the FA composition of HM, collected from mothers with different health conditions (normal Body Mass Index (nBMI); overweight and obese) using GC-FID method. The results showed that saturated FAs were present in the highest amount in the HM samples, of which palmitic acid was the main representative. The major monounsaturated FA was oleic acid, while linoleic acid was the most abundant of the polyunsaturated FAs (PUFA). Overweight and obese women have lower levels of PUFA in their breast milk. The data were subjected to principal component and quadratic discriminant analysis (QDA). QDA classified nBMI and overweight and obese mother milk samples with 88.24% accuracy. Significant differences were found between normal and overweight and obese HM samples in case of C10:0 and C18:3 FAs. Higher maternal BMI was associated with a higher n-6/n-3 PUFA ratio.