Objective To identify genotypes and potential routes of dissemination of P.aeruginosa isolates in Beijing using Multilocus-locus variable-number tandem-repeat(VNTR) analysis (MLVA). Methods One hundred and forty one experimental animals and facilities isolates of P.aeruginosa were detected by selected 13 VNTRs loci. Then we draw the phylogenetic tree and minimum spanning tree(MST) using BioNumerics software according to fingerprint clustering analysis. Results All isolates of P. aeruginosa was divided into three major genetic Clusters, 56 genotypes. The proportion of A cluster is 82.3%, B cluster 12.8%, C cluster 5.0%. The Simpson’s index of diversity is 0.763. The isolates have distant homology in the neighboring facilities. ConclusionsMLVA displayed excellent discriminatory power for P.aeruginosa. MLVA can effectively track P.aeruginosa source. The P. aeruginosa isolates of experimental animals have rich polymorphism genotypes and no regional homology in Beijing.