Abstract: Virus was isolated from scab materials of lambs from a goatfarm in Qingyuan city of Guangdong province with clinical sore mouth symptom by passaging in MDBK cells, A strain of Orf virus was successfully isolated and identified, named ORFV/GD-QY/01. A pair of specific primers were designed according to B2L gene sequence in NCBI, and PCR method was used to amplify the B2L gene. Phylogenetic analysis of major envelope B2L gene of parapoxvirus with DNAStar. The result showed that MDBK cell cultures infected with crust materials displayed cytopathic effects, B2L gene was amplified，the isolated strain was closest to similarity of nucleotide sequences（99.2%） with those of Taiwan strain（EU93510 6, DQ904351）.