(1,3;1,4)-beta-D-glucans consist of unbranched and unsubstituted chains of (1,3)- and (1,4)-beta-glucosyl residues, in which the ratio of (1,4)-beta-D-glucosyl residues to (1,3)-beta-D-glucosyl residues appears to influence not only the physicochemical properties of the polysaccharide and therefore its functional properties in cell walls, but also its adoption by different plant species during evolution. The (1,3;1,4)-beta-D-glucans are widely distributed as non-cellulosic matrix phase polysaccharides in cell walls of the Poaceae, which evolved relatively recently and consist of the grasses and commercially important cereal species, but they are less commonly found in lower vascular plants, such as the horsetails, in algae and in fungi. The (1,3;1,4)-beta-D-glucans have often been considered to be components mainly of primary cell walls, but recent observations indicate that they can also be located in secondary walls of certain tissues. Enzymes involved in the depolymerisation of (1,3;1,4)-beta-D-glucans have been well characterized. In contrast, initial difficulties in purifying the enzymes responsible for (1,3;1,4)-beta-D-glucan biosynthesis slowed progress in the identification of the genes that encode (1,3;1,4)-beta-D-glucan synthases, but emerging comparative genomics and associated techniques have allowed at least some of the genes that contribute to (1,3;1,4)-beta-D-glucan synthesis in the Poaceae to be identified. Whether similar genes and enzymes also mediate (1,3;1,4)-beta-D-glucan biosynthesis in lower plants and fungi is not yet known. Here, we compare the different fine structures of (1,3;1,4)-beta-D-glucans across the plant kingdom, present current information on the genes that have been implicated recently in their biosynthesis, and consider aspects of the cell biology of (1,3;1,4)-beta-D-glucan biosynthesis in the Poaceae.