Pseudomonas putida is a bacterium commonly found in soils, water and plants. Although P. putida group strains are considered to have low virulence, several nosocomial isolates with carbapenem- or multidrug-resistance have recently been reported. In the present study, we developed a multilocus sequence typing (MLST) scheme for P. putida. MLST loci and primers were selected and designed using the genomic information of 86 clinical isolates sequenced in this study as well as the sequences of 20 isolates previously reported. The genomes were categorised into 68 sequence types (STs). Significant linkage disequilibrium was detected for the 68 STs, indicating that the P. putida isolates are clonal. The MLST tree was similar to the haplotype network tree based on single nucleotide morphisms, demonstrating that our MLST scheme reflects the genetic diversity of P. putida group isolated from both clinical and environmental sites.