Glucosylceramide (GluCer) is a major sphingolipid of plant tissue and, thus, abundant in nature and in dietary food sources. The lipid backbones of mammalian GluCer (sphingosine, d18 : 1^Δ4, and ceramide) induce cell death (apoptosis) and inhibit colon carcinogenesis, it is critical to know the structures of GluCer present in plants as a first step toward understanding this potential link between diet and cancer. This study characterized the molecular species of GluCer from soybean and wheat by low‐resolution, high‐resolution and tandem mass spectrometry. Soybean GluCer was comprised primarily (>95%) of ceramide with 4,8‐sphingadiene (d18 : 2^Δ4,Δ8) and α‐hydroxypalmitic acid (h16 : 0); the remainder had the same backbone with h18 : 0, h20 : 0, h22 : 0 and h24 : 0 fatty acids. Wheat GluCer had three major ceramide, d18 : 2^Δ4,Δ8 with h16 : 0, d18 : 1^Dgr;8 with h16 : 0 and d18 : 2^Δ4,Δ8 with h20 : 0, and smaller amounts of other homologs. These backbones differ from those of mammalian sphingolipids, which often have a Δ4‐double bond (but rarely a Δ8‐double bond), and have α‐hydroxy fatty acids in only some cases. Previously unexplained fragmentations that were diagnostic for the type of sphingoid base backbone (i.e. by homolytic cleavage of the doubly allylic C‐6—C‐7 bond to yield a stable distonic allylic radical cation and an allylic radical neutral) were also identified. Hence this method should be useful in the identification of double bonds in sphingolipids, and structure–function relationships between sphingolipids and colon carcinogenesis. Copyright © 2000 John Wiley & Sons, Ltd.