Balance diet is a basic factor in livestock productivity. Uneven and unbalanced dietary pattern is considered a major restriction for livestock development in Pakistan because it leads to decrease milk production, reproductive performance and growth rate of cattle. Livestock productivity can be increases by probiotics supplementation in cattle feed because they have ability to survive harsh gut environment, enhance digestion, improve microbial balance inside the gut and subsequently enhance milk production and growth rate. Cattle gut is rich source of probiotic species. There is still insufficient knowledge on indigenous probiotic species of livestock gastrointestinal tract. The present experiment was conducted to isolate and identify probiotic bacterial species from cattle gut at molecular level and to determine their probiotic potential. 35 pure bacterial strains were isolated from 30 fecal samples of healthy lactating Sahiwal cow. These strains were identified on the basis of morphological (colony morphology and gram staining), biochemical (catalase and oxidase test) and molecular characterization. Three strains (PBC-3, PBC-6 and PBC-9) were selected as probiotic strains based on their enzymatic potential (cellulolytic, amylolytic and proteolytic ability) and survival ability to low pH. These isolates were identified as Enterococcus faecalis (PBC-3), Pediococcus acidilacticii (PBC-6) and Pediococcus pentosaceus (PBC-9) by 16S rRNA gene sequencing. Results of present study illustrated that these three bacterial strains (Enterococcus faecalis , Pediococcus acidilacticii and Pediococcus pentosaceus) showed best probiotic potential and can be use in cattle feed in future.