Abstract: Objective To analyze the molecular characteristics of measles virus of genotype B3 isolated firstly in Guizhou province in 2014.Methods Throat swab specimens were collected from patients with clinically diagnosed measles and tested for viral RNA using real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) after RNA extraction.Fragments of 634 nucleotides of the carboxyl end of nucleoprotein gene were amplified by using reverse transcription-polymerase chain reaction(RT-PCR) and then the PCR products were directly sequenced and analyzed.The phylogenetic analysis was based on the 450 bp C-terminal end of the N gene sequences.Results The isolated virus strain, named MVs/Guizhou.CHN/11.14/01, was clustered within the same genotype group with World Health Organization(WHO) genotype B3 reference strain, Ibadan.NGA/0.97/1(AJ232203).The nucleotide and amino acid divergence were 2.00% and 2.67% compared with Ibadan.NGA/0.97/1(AJ232203).Compared with WHO H1 genotype reference strain(MVi/Hunan.CHN/0.93/7), the nucleotide and amino acid divergence were 10.8% and 12.0%, respectively.The evolutionary distance of nucleotide and amino acid of the B3 genotype and the representative WHO 22 genotype strains were between 4.40%-11.0% and 4.67%-12.0%.Conclusion A new genotype B3 measles virus was isolated in Guizhou province in 2014 and the identification of the virus strain is helpful to control and eliminate measles epidemic in Guizhou province.