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The 16S-23S rRNA gene internal transcribed spacer (ITS) regions of Klebsiella spp., including Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena , and Klebsiella ornithinolytica , were characterized, and the feasibility of using ITS sequences to discriminate Klebsiella species and subspecies was explored. A total of 336 ITS sequences from 21 representative strains and 11 clinical isolates of Klebsiella were sequenced and analyzed. Three distinct ITS types—ITS none (without tRNA genes), ITS glu [with a tRNA Glu (UUC) gene], and ITS ile+ala [with tRNA Ile (GAU) and tRNA Ala (UGC) genes]—were detected in all species except for K. pneumoniae subsp. rhinoscleromatis , which has only ITS glu and ITS ile+ala . The presence of ITS none in Enterobacteriaceae had never been reported before. Both the length and the sequence of each ITS type are highly conserved within the species, with identity levels from 0.961 to 1.000 for ITS none , from 0.967 to 1.000 for ITS glu , and from 0.968 to 1.000 for ITS ile+ala . Interspecies sequence identities range from 0.775 to 0.989 for ITS none , from 0.798 to 0.997 for ITS glu , and from 0.712 to 0.985 for ITS ile+ala . Regions with significant interspecies variations but low intraspecies polymorphisms were identified; these may be targeted in the design of probes for the identification of Klebsiella to the species level. Phylogenetic analysis based on ITS regions reveals the relationships among Klebsiella species similarly to that based on 16S rRNA genes.