Plant carbohydrates are of increasing interest as renewable feedstocks to replace petrochemicals in the generation of fuels and production of high-value chemicals. Greater understanding of the genetic control of diversity in fructan synthesis and accumulation would facilitate more directed channelling of feedstock to process in a ryegrass biorefinery. Divergent populations produced by phenotypic selection for water-soluble carbohydrate content have been used to investigate relationships between traits, and to identify patterns of genetic differentiation which indicate genomic regions under high and low selection pressure. Selection for high water-soluble carbohydrate content was associated with increased synthesis of large fructan polymers and increased accumulation of above-ground plant biomass, particularly during spring. Three rounds of selection and two rounds of recombination resulted in widespread genetic differentiation across the whole genome, causing reduced allelic richness and increasing homozygosity at some loci. A smaller number of loci were shown to be subject to high selection pressure. Breeding material subjected to many years of selection for water-soluble carbohydrate also showed allelic differences which may reflect the consequences of high selection pressure at some of these same loci. However, some of the loci unaffected in the divergent selection experiment showed similar effects. This might arise from differences in linkage disequilibrium in these two sets of plant materials, but more likely from the different genetic background of the germplasm. This illustrates the complex nature of the water-soluble carbohydrate trait in perennial ryegrass.