Abstract: Outbreaks of ORF virus (ORFV) infection in small ruminants, which occurred in eight districts of Karnataka, India were investigated during this study. Seventy scab samples were collected from affected sheep and goats from 27 outbreaks and subjected for virus isolation and polymerase chain reaction. The ORFV was successfully isolated in the chicken embryos (CE) by CAM route of inoculation. Polymerase chain reaction (PCR) was standardized targeting B2L gene for detection of ORFV. The screening of 70 samples for B2L gene amplification revealed 60 per cent positivity of Orf infection. The phylogenetic analysis based on nucleotide and deduced amino acid sequences of full length and partial B2L gene revealed that, the ORFV isolates had highest genetic homology among them and with the other Indian ORFV isolates. The maximum genetic heterogeneity was observed with ORFV isolates from other countries. Further, full length B2L gene sequence based analysis provided significant information about the genetic relationship among the various ORFV isolates. Sequence analysis revealed high nucleotide (98.7 -100 per cent) and amino acids (98.4-99.7 per cent) identity with Indian isolates maximum with Assam/09 and Assam/10 ORFV isolates.