Abstract: Considering the importance of polycentric anaerobic rumen fungi in fiber digestibility and in the paucity of any reports in Indian cattle an attempt was made to isolate and characterize Orpinomyces species employing a combination of both morphological and molecular methods. Around six hundred rumen liquor/fecal samples of cows and buffaloes were collected from various locations within the country in order to characterize Opinomyces species predominant in Indian cattle and buffaloes using medium 10X and hungate roll tubes. Orpinomyces genera were characterized by the presence of numerous coralloid sporangiophore complexes with large bulbous sporangia and polyflagellated zoospores similar to those of Neocallimastix sp., Orpinomyces joyonii and Orpinomyces intercalaris were the two predominant species obtained with the former being characterized by the spherical sporangia developing on branched or simple sporangiophore complexes with the hypae having light constrictions, while the latter had globose sporangia developing from expansion of hyphae or as lateral outgrowths but rarely terminal. The hydrolytic enzyme activities of the two species of Orpinomyces showed that O. joyonii was characterized by high endoglucanase, xylanase and β-glucosidase activity while Orpinomyces intercalaris showed higher activities of cellobiohydrolase and β-xylosidase showing different strategy for fiber degradation. The DNA-based methodologies were used to identify the isolated Orpinomyces using Internal transcribed spacer region (ITS1) analysis of the fungal rDNA genes. Three of the Indian isolates (NIANP 49, NIANP 58 and NIANP 60) had motifs 5, 8, 7 and 5 corresponding to Variable region I, II, III and IV, respectively and morphologically were identified as Orpinomyces joyonii. The sequence of two isolates (NIANP 57 and 59) however, was different from the rest of the isolates in having motif 8 in Variable region III and morphologically were identified as Orpinomyces intercalaris. Multiple alignments of obtained sequences showed all the isolates to fall within a single group together with the Piromyces II group. By the combination of morphological and phylogenetic analysis we showed that gut of Indian cattle and buffaloes was predominated by only two species viz.Orpinomyces joyonii and Orpinomyces intercalaris.