The experiments reported in this study provided a more comprehensive insight into the effect of chemical composition on the crystallization behavior of milk fat (MF). MF was fractionated between 20 and 40°C into nine fractions with different melting points and was first subjected to the heating step (L20, L30, L40, and S40) followed by the cooling phase (SS40, SL40, SS30, SL30, and LL40). Furthermore, the species of fatty acids (FAs) and triglycerides (TAGs) of the MF fractions were identified. The thermodynamics, crystallization behavior, and polymorphs were determined using differential scanning calorimetry, pulsed nuclear magnetic resonance, and X-ray diffraction, respectively. The results indicated that L40 yielded the highest percentage (ß35% of the total MF) of all the fractions. Enthalpies of the melting and crystallization processes of solid fat content in this study were related to the different FA and TAG compositions of MF and its fractions. High melting fractions (HMFs) were enriched with long-chain saturated fatty acids and tri-saturated (S3) TAGs, and low melting fractions (LMFs) were enriched with short-chain unsaturated FAs and tri-unsaturated (U3) TAGs. Moreover, the various nucleation mechanisms of MF fractions were identified according to the Avrami equation. The polymorphic transformation from a β' form of double chain length structures to a β form of triple chain length occurred in the native MF and HMFs, whereas the LMFs displayed almost no crystals.Practical Application: This study represented the first time that nine fractions were obtained using MF fractionation via a heating step, followed by a cooling phase. Furthermore, the chemical composition of MF fractions was investigated. The results obtained from this study might be of specific value in understanding the functional properties of fat-based dairy food in both storage conditions and real-time applications.