India is the largest buffalo milk producer in the world. Buffalo milk is rich in minerals like calcium and magnesium, contains 100% more fat and less cholesterol than cow milk. Physiochemical properties of dairy products depend upon the size and distribution of milk fat globules (MFG). Individual MFG from Murrah buffalo were analyzed as a function of size and distribution and comparisons were made with MFG of crossbred (Holstein Friesian × Sahiwal) cows. Main objectives of this study was to measure areas of buffalo MFG and analyze percentage of crescents associated with MFG using a simple microscopic technique. Crescents of MFG can provide an alternative source of RNA of mammary epithelial cells origin. Acridine orange staining of fresh milk was visualized by light microscopy under red and green channel filters. MFG and MFG with crescents were analyzed using ImageJ image analysis software. The percentage of large sized MFG (area >20 µm²) was greater and the percentage of small sized MFG (area <10 µm²) was lower in buffalo than cow milk. The percentage of crescents was < 1% of total MFG in both the species. These results indicate that MFG in buffalo milk is larger than that MFG of cow milk. RNA isolation from buffalo milk fat and its suitability for microarray analysis remains to be determined.