Objective: The 3×3 factorial arrangement was used to investigate if either high water-soluble carbohydrates (WSC) cultivars or suitable time of day that the grass cut could improve nutrient values and in vitro fermentation characteristics. Methods: The 3 cultivars were mowed at 3 diurnal time points and included a benchmark WSC ryegrass cultivar 'Premium', and 2 high WSC cultivars AberAvon and AberMagic, which contained, on average, 157, 173, and 193 g/kg dry matter (DM) of WSC, and 36.0, 36.5, and 34.1 g/kg DM of N during 7th regrowth stage, respectively. The fermentation jars were run at 39℃ with gas production recorded and sampled at 2, 5, 8, 11, 14, 17, 22, 28, 36, and 48 h. The rumen liquid was collected from 3 rumen fistulated cows grazing on ryegrass pasture. Results: High WSC cultivars had significantly greater WSC content, in vitro DM digestibility (IVDMD) and total gas production (TGP), and lower lag time than Premium cultivar. Methane production for AberMagic cultivar containing lower N concentration was marginally lower than that for AberAvon and Premium cultivars. Grass cut at Noon or PM contained greater WSC concentration, IVDMD and TGP, and lower N and neutral detergent fiber (NDF) contents, but CH4 production was also increased, compared to grass cut in AM. Meanwhile, the effects of diurnal cutting time were influenced by cultivars, such as in vitro CH4 production for AberMagic was not affected by cutting time. The IVDMD and gas production per unit of DM incubated were positively related to WSC concentration, WSC/N and WSC/NDF, respectively, and negatively related to N and NDF concentrations. Conclusion: These results imply either grass cut in Noon or PM or high WSC cultivars could improve nutrient values, IVDMD and in vitro TGP, and that AberMagic cultivar has a slightly lower CH4 production compared to AberAvon and Premium. Further study is necessary to determine whether the increase of CH4 production response incurred by shifting from AM cutting to Noon and/or PM cutting could be compensated for by high daily gain from increased WSC concentration and DM digestibility.