Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These show not only the excellent surface-active properties but also versatile biochemical actions. In course of MEL production from soybean oil by P. shanxiensis, new extracellular glycolipids (more hydrophilic than the previously reported MELs) were found in the culture medium. As a result of the structural characterization, the glycolipids were identified as a mixture of 4-O-[(2′, 4′-di-O-acetyl-3′-O-alka(e)noyl)-β-D-mannopyranosyl]-D-erythritol and 4-O-[(4′-O-acetyl-3′-O-alka(e)noyl-2′-O-butanoyl)-β-D-mannopyranosyl]-D-erythritol. Interestingly, the new MELs possessed a much shorter chain (C2 or C4) at the C-2′ position of the mannose moiety compared to the MELs hitherto reported, which mainly possess a medium-chain acid (C10) at the position. They would thus show higher hydrophilicity and/or water-solubility, and expand the development of the environmentally advanced yeast biosurfactants.