This study assessed the effect of heat treatment on the physical properties of bovine milk samples possesing different β-casein (β-CN) phenotypes. After heat treatment at 72 °C/15 s, 121 °C/2.6 min, or 140 °C/3 s, β-CN milks were analysed using Fourier Transform Infrared (FTIR) spectroscopy, Nuclear Magnetic (1H NMR) resonance, and a number of physicochemical measurements. Principal Component Analysis (PCA) was used to provide a discrimination of samples. In contrast to significant amounts of intramolecular β-sheet, β-turn, and random coil, in A1/A1, A1/A2, and A2/A2 β-CN milks, respectively, increasing the heat treatment temperature decreased the level of intramolecular β-sheets in all three types of bovine milk. The main difference involved a higher presence of aggregated β-sheet structures in A1/A2 β-CN milk likely due to the presence of tyrosine. A1/A1 and A1/A2 β-CN milks were characterised with greater amounts of calcium and phosphorus, and a higher net negative zeta potential than A2/A2 β-CN milk. Furthermore, A2/A2 β-CN milk was composed of larger casein micelle particles with lower levels of κ-CN compared to the other β-CN milk phenotypes. These findings may assist in predicting the behaviour of β-CN milks during relevant industrial processing.