In this study, the nucleotide sequences of the complete leader proteinase (Lpro) region of 21 isolates of foot-and-mouth disease virus (FMDV) serotype O collected during various outbreaks in India were sequenced and compared with vaccine strains. The phylogenetic analysis of these Lpro sequences showed a difference in the clustering of the isolates based on the VP1 capsid coding region sequences. The comparison of amino acid sequences at the N terminus end of the Lpro region showed very high variability, although 2 conserved start codons (AUG) at 1st and 29th sites. Furthermore, all the amino acid residues that formed the active cleft site of the Lpro sequences of this study were conserved. These results suggest that Lpro sequences could also be used for phylogenetic comparison of FMDV isolates.