Pseudomonas putidais a major species belonging to the genusPseudomonas. Although several hundred strains ofP. putidahave been deposited in culture collections, they potentially differ from the genetically defined “truePseudomonas putida” because many were classified asP. putidabased on their phenotypic and metabolic characteristics. A phylogenetic ana­lysis based on the concatenated sequences of the 16S rRNA andrpoDgenes revealed that 46 strains ofP. putidadeposited in Japanese culture collections were classified into nine operational taxonomic units (OTUs) and eleven singletons. The OTU7 strain producesN-acylhomoserine lactone as a quorum-sensing signal. One of the OTU7 strains, JCM 20066, exhibiteda ppuI-rsaL-ppuRquorum-sensing system that controls biofilm formation and motility. TheP. putidatype strain JCM 13063Tand six other strains were classified as OTU4. Classification based on the calculation of whole-genome similarity revealed that three OTU4 strains, JCM 20005, 21368, and 13061, were regarded as the same species as JCM 13063Tand defined as trueP. putida. When orthologous genes in the whole-genome sequences of trueP. putidastrains were screened, PP4_28660 fromP. putidaNBRC 14164T(=JCM 13063T) was present in all trueP. putidagenome sequences. The internal region of PP4_28660 was successfully amplified from all trueP. putidastrains using the specific primers designed in this study.