In vitro gas production technique (IVGPT) is a routine method in nutritional sciences to determine energy content, organic matter (OM) digestibility, and fermentation kinetics of feedstuffs. After collecting from two ruminally cannulated Holstein heifers (350 kg), rumen liquors were used either fresh or cryopreserved form in the inoculums for IVGPT. Starch- (barley, wheat, and corn) and protein-rich (sunflower meal, cotton seed meal, and soybean meal) feedstuffs were evaluated for gas production kinetics, fermentation pattern, and energy content in 5 replicates. pH, NH3-N concentration and volatile fatty acids (VFA) profile, gas production, and fermentation kinetics parameters were measured. Data were analyzed by 2-way ANOVA. Viable protozoa rate was found to be 70.8% in cryopreserved rumen liquor after thawing. Decrease in pH in thawed rumen liquor was less than fresh rumen liquor as the incubation period advanced. Utilization of frozen rumen liquor after thawing in IVGPT was associated with lower VFA and NH3-N concentration, cumulative gas production, and metabolisable energy estimate for all feedstuffs. In conclusion, despite high correlation between in vitro data obtained from fresh and thawed rumen liquors to predict gas production, further experiments should cope with improving cryopreservation protocol for rumen liquors in order to optimize microbial activity for maintaining fermentation pattern.