The effect of cream heat treatment prior to butter manufacturing, fluctuating temperatures during storage and presence of fat globules vs. no fat globules was examined in laboratory scale produced butter. X-ray diffraction and differential scanning calorimetry was used to study crystallization behaviour and nuclear magnetic resonance to measure solid fat content and water droplet size distribution. Furthermore, the crystal structure was linked to the rheological properties and microstructure of the butter using confocal laser scanning microscopy. Butter produced from non-matured cream mainly formed α- and β′-crystals with minor traces of β-crystals. Maturing of the cream caused a transition from α- to β′- and β-form. The rheological behaviour of slow cooled butter deviated from the matured ones by having a lower elastic modulus, caused by a weaker crystal network. Presence of fat globules did not affect the rheological properties significantly.