The grain of rye (Secale cereale L.) used for baking contains a large amount of non-starch polysaccharides, making it an excellent component of functional foods. But rye grain intended for alcohol production and forage use should have a reduced content of these polysaccharides. A comprehensive parameter that can predict the best field of application for winter rye grain is the viscosity of its wholemeal water extract. However, our understanding of the genetic background underlying this key trait and associated features of rye grain is poor. By analyzing six Russian winter rye cultivars, we identified the most contrasting forms and characterized the peculiarities of their water-soluble carbohydrates capable of influencing the viscosity of water extracts. Then, using phylogenetic and transcriptomic analyses, we identified in the rye genome many genes encoding putative glycosyltransferases and glycosylhydrolases responsible for the synthesis and degradation of arabinoxylans, mixed-linkage glucans, cellulose, and some other polysaccharides. We determined the dynamics of mRNA abundance for these genes at three stages of kernel development. Comparisons of gene expression levels in two contrasting cultivars revealed specific members of multigene families that may serve as promising targets for manipulating non-starch polysaccharide content in rye grain. High-viscosity cultivars were characterized by up-regulation of many glycosyltransferases involved in the biosynthesis of arabinoxylans and other cell-wall polysaccharides, whereas low-viscosity cultivars showed up-regulation of several genes encoding polysaccharide-degrading enzymes.