Objective: The aim of the present study was isolation and characterization of phospholipids from eri pupal oils. Methods: Silkworm lipids were extracted from pupae fed on castor and tapioca leaves and further separated in to individual class of lipids by column chromatography. Fatty acid composition of each lipid class was studied along with positional distribution of fatty acids in major phospholipids by enzymatic hydrolysis. Results and Conclusion: The eri pupal oils were found to contain 96.72-97.33% of neutral lipids, 0.51-0.21% of glycolipids and 2.65-2.39% phospholipids. Phosphatidylethanolamine (PE) was the predominant classes of phospholipid present (65.65 and 64.29%) in tapioca and castor fed pupal oils respectively. Phosphatidylcholine (PC) was observed in 19.50 and 19.22% respectively in tapioca and castor fed pupal oils. Cardiolipin (CL) and phosphatidylinositol (PI) are observed in minor levels. Positional distribution of fatty acids showed that, __ampersandsignalpha;-linolenic acid (ALA) was found to be more in sn-2 position of PE. Thus pupal oil offers a good natural source of PE with enriched ALA.