Two varieties each of spelt (Triticum aestivum var. spelta), durum wheat (Triticum turgidumvar. durum), rye (Secale cereale), barley (Hordeum vulgare), oats (Avena sativa), einkorn (Triticum monococcum var. monococcum) and emmer (Triticum turgidum var. dicoccum) (all members of the Pooideae sub-family of grasses) were selected according to variation in their contents of soluble and/or total arabinoxylan (AX) determined during the HEALTHGRAIN diversity screen, together with one genotype of the related “model” grass species Brachypodium distachyon. The spatial distribution of low substituted (LS-AX) and highly substituted arabinoxylan (HS-AX) was determined using FT-IR spectroscopic mapping of transverse thin cross-sections consisting of cell walls only. Variation in cell wall AX composition was observed between the cereals, and compared with that observed for wheat (Triticum aestivum var. aestivum). One line of each cereal type was analysed in more detail using 1H NMR spectroscopy. The results of the two analyses were consistent, showing variation in the composition and structure of the endosperm cell wall AX that is consistent with the genetic relationships of the cereals studied.