Sequence analysis of segment 2 (seg-2) of three Indian bluetongue virus (BTV) isolates, Dehradun, Rahuri and Bangalore revealed 99% nucleotide identity amongst them and 96% with the reference BTV 23. Phylogenetic analysis grouped the isolates in 'nucleotype D'. The deduced amino acid (aa) sequence of the Bangalore isolate showed a high variability in a few places compared to other isolates. B-cell epitope analyses predicted an epitope that is present exclusively in the Bangalore isolate. Two-way cross serum neutralization confirmed that Bangalore isolate is antigenically different from the other two isolates. The results of this study suggest that these three isolates are VP2 variants of BTV 23. This signifies that non-cross-neutralizing variants of the same BTV serotype should be included in vaccine preparation.