Native fat globules composed mainly of triglycerides are secreted as droplets of variable size. The size of fat globules affects the form of fat in dairy products and final functionality, which depends mainly on the composition of the globules and membrane. However, the relation between the composition and size of fat globules has not been studied in detail. In this study, differences in the lipid content and fatty acid composition related to the size of native fat globules were investigated using confocal Raman spectroscopy, which offers the possibility of acquisition and analysis of the Raman signal without disruption of a single fat globule in natural suspension. The results showed small fat globules (SFG) had a higher ratio of band intensities at 2 885/2 850 cm-1, indicating SFG tend to have a triglyceride core in a fluid state with a milk fat globule membrane in a crystalline state. In addition, the SFG had a higher level of unsaturation compared to large fat globules, shown by a lower ratio at 1 655/1 443 cm-1. Using cream with selected SFG would allow a harder and more costly churning process but lead to a softer butter.