Endosperm cell walls of cultivars of wheat (Triticum aestivum L.) selected for their endosperm texture (two soft and two hard) were analysed in situ by Fourier transform infrared (FTIR) microspectroscopy. FTIR imaging coupled with statistical analysis was used to map the compositional and structural heterogeneity within transverse sections from which cell contents had been removed by sonication. In the majority of grains analysed, two distinct populations of endosperm cells could be identified by spectral features that were related to cell morphology and age, regardless of cultivar. The main cell-wall component responsible for these differences was the polysaccharide arabinoxylan. In a few samples, this heterogeneity was absent, for reasons that are not understood, but this was not correlated to endosperm texture or growth conditions. Within the same population of endosperm cells, cell walls of hard endosperm could be distinguished from those of soft endosperm by their spectral features. Compared to hard cultivars, the peripheral endosperm of soft cultivars was characterised by a higher amount of polymer, whose spectral feature was similar to water-extractable arabinoxylan. In contrast, no specific compound has been identified in the central endosperm: structural differences within the polysaccharides probably contribute to the distinction between hard and soft cultivars. In developing grain, a clear difference in the composition of the endosperm cell walls of hard and soft wheat cultivars was observed as early as 15 days after anthesis.