In this study, we characterized the polymorphism of milk fat (MF) with various TAG compositions during isothermal crystallization at 20 °C. TAG composition of MF from seven individual cows was determined using GC-FID and MALDI-TOF MS, and MF polymorphism was studied using X-ray diffraction. Results showed that TAG profile determines the polymorphic behavior of MF. Saturated TAG with carbon numbers 34-38 promoted the formation of α polymorphs, whereas unsaturated TAG with 52-54 promoted the formation of the β polymorphs. Furthermore, MFs with unsaturated fatty acid profiles were increased in unsaturated TAG with 52-54 carbons. The presence of MF crystals in the β polymorph has been controversial over the years as most authors mainly find MF crystals in the α and β′ form. In our work, we showed that the β polymorph is formed in MF on the basis of its TAG composition.