Perennial ryegrass (Lolium perenne L.) cultivars have been developed to express higher levels of leaf water-soluble carbohydrates (WSC), but expression of this high sugar' trait varies between environments and is likely to be further influenced by the extent of plant re-growth. The herbage WSC concentration and the ratio of WSC to crude protein (WSC : CP) in high sugar cultivars AberMagic and SF Joule were therefore compared with a control cultivar, Arrow, under cool temperate Tasmanian conditions and two defoliation interval treatments. The irrigated cultivars were subjected to defoliation at either the 15-leaf or 3-leaf stage of re-growth, and additional components of nutritive value (fibre concentrations and metabolizable energy content) and dry matter (DM) yields were measured throughout a 12-month period (March 2011 to March 2012). The high sugar trait was consistently expressed in AberMagic, which under both the 15-leaf and 3-leaf stages defoliation intervals, displayed the highest WSC concentration (mean 194 and 247 mg/g DM, respectively, compared with 153 and 178 mg/g DM for Arrow) and highest WSC : CP ratio (mean 074 and 129, respectively, compared with 058 and 085 for Arrow). Defoliation at the 3-leaf stage of regrowth ensured greater expression of the high sugar trait in both AberMagic and SF Joule, as measured by the increase in WSC concentration and WSC : CP ratio as a result of increasing defoliation interval. The strength and consistency of trait expression in AberMagic under the 3-leaf stage defoliation interval warrants further research to investigate its effect on rumen nitrogen (N) partitioning and milk production in this environment.