Tetraacylglycerol (an acylglycerol estolide) contains an acyl chain attached to the hydroxyl group of another acyl chain attached to the glycerol backbone. Lesquerolic acid (Ls, OH1420:111) is the main fatty acid in lesquerella oil and may be used industrially for the manufacture of biodegradable industrial products. Electrospray ionization mass spectrometry of the lithium adducts of acylglycerols in the high-performance liquid chromatography fractions from the seed oil of lesquerella (Physaria fendleri) was used to identify thirteen tetraacylglycerols. They were LsLsLsLn, LsLsLsL, LsLs-OH20:2-O, LsLsLsO, LsLsLnLn, LsLsLLn, LsLsOLn, LsLsLL, LsLsOL, LsLsOP, LsLsOO, LsLsLS and LsLsOS. The OH20:2 was auricolic acid (OH1420:211,17). For the four tetraacylglycerols containing one normal fatty acid (non-hydroxy fatty acid), LsLsLsLn, LsLsLsL, LsLs-OH20:2-O and LsLsLsO, the normal fatty acids were all directly attached to the glycerol backbone, not to the hydroxyl group of fatty acids. We propose that the biosynthetic precursors (triacylglycerol acyltransferase) of these four tetraacylglycerols were LsLsLn, LsLsL, LsLsO (Ls-OH20:2-O) and LsLsO individually. LsLsO and Ls-OH20:2-O were equally active as the biosynthetic precursors for LsLs-OH20:2-O. For LsLsLS, linoleate were all attached to the glycerol backbone and LsLsL was proposed to be the biosynthetic precursor. For LsLsOS, stearate were all attached to the glycerol backbone and LsLsS was proposed to be the biosynthetic precursor. For the other seven tetraacylglycerols containing two normal fatty acids, LsLsAB, the biosynthetic precursors could be both LsLsA and LsLsB.