Objective: The 3×3 factorial arrangement was used to investigate if either high watersoluble carbohydrates (WSC) cultivars or suitable time of day that the grass cut could improve nutrient values and <i>in vitro</i> 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°C 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, <i>in vitro</i> 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 <i>in vitro</i> 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 <i>in vitro</i> 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.