The present study describes characterization of a novel rotavirus (B29) isolated from a buffalo calf causing severe diarrhea. It was confirmed by VP4 and VP7 gene based RT-PCR and in sequence analysis, the VP4 gene showed up to 99.3% sequence identity at nucleotide as well as amino acid levels with P[3] rotavirus genotypes. Similarly, VP7 gene showed a maximum nucleotide and amino acid identity of 99.1 and 98.7%, respectively with G3 genotypes of group A rotavirus (RVA) from several host species and places. The phylogenetic analysis of VP4 gene also revealed close relatedness with other P[3] genotype of rotaviruses from bovine, goat, canine and feline origin. Similarly, VP7 gene revealed close relation with several G3 rotaviruses from bovine and equine origin. The buffalo rotavirus isolate B29 was genotyped as G3P [3]. This is the first report of rotavirus isolate with a G3P[3] genotypic constellation in buffalo calvesfrom this region of India. The detection of buffalo rotavirus of G3P[3] genotypic combination reveals an unexpected epidemiological situation and diversity of bovine rotaviruses in India.