Abstract: The inclusion of yeast (Saccharomyces cerevisiae) in animal feeds as a probiotic is well recognized in the livestock industry, however, the effects on animal performance is inconsistent and depends on the type of strain used and the ability to be viable in the gut. Recently, we have isolated a stress-resistant yeast culture strain (OBV9) to tolerate and be effective in the rumen environment. The objectives of this study were (1) To determine the concentration of the optimum level of yeast to be included in the animal feed and (2) To evaluate the effect of total mixed rations with and without the supplementation of two different (mesophilic and stress-resistant) strains of yeast (Saccharomyces cerevisiae) on the productive performance of lactating murrah buffaloes. An in vitro anaerobic fermentation study was carried out with 0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 g of the yeast culture strain OBV9 using both Tilley and Terry method and also Menke’s in vitro gas production method. Both methods indicated that 1.0 g kg–1 concentration of yeast culture favored microbial biomass production, DM digestibility and also in vitro gas production. A subsequent in vivo experiment was conducted for 180 day with 18 lactating buffaloes (Murrah breed) which were randomly assigned to three treatment groups based on yeast culture supplementation (diet 1-control, diet 2-mesophilic yeast culture and diet 3-stress-resistant yeast culture). Measurements on voluntary feed intake and milk composition were recorded daily. Samples for metabolic measurements were taken over a fifteen day period towards the end of the experiment. Results indicated that stress-resistant yeast culture supplementation increased the digestibilities of dry matter, hemicellulose and cellulose (p<0.05). Although, the total milk production remained similar between the diets, supplementation of yeast culture increased both the milk protein and somatic cell counts (p<0.05), while stress-resistant yeast culture also increased fat content in the milk (p<0.06). It is concluded that stress-resistant yeast supplementation at the rate of 1 g kg–1 of DM to lactating buffaloes had a positive impact on milk production.