A comparative analysis of fatty acid contents between ultra-high temperature (UHT) milk and reconstituted milk was carried out using 1H nuclear magnetic resonance (1H NMR) spectroscopy. The results showed that the relative contents of linolenic acid, linoleic acid, oleic acid and saturated fatty acid in UHT milk and reconstituted milk were (0.98 ± 0.11)% and (0.90 ± 0.04)%, (1.15 ± 0.36)% and (1.11 ± 0.36)%, (27.30 ± 0.56)% and (26.78 ± 0.78)%, and (43.26 ± 1.56)% and (43.81 ± 2.34)%, respectively. Orthogonal partial least squares discriminant (OPLS-DA) analysis and independent sample t test were performed on the 1H NMR spectrum peaks and fatty acid contents of the two milks. It was found that the relative content of linolenic acid was significantly different (P < 0.05) between the two milks. Nutritional assessment showed that UHT milk was more suitable than reconstituted milk for people with hyperlipidemia and/or high cholesterol, while the nutritional value of other fatty acids was similar between both milks.