b Klebsiella pneumoniae, a Gram-negative bacillus that causes life-threatening infections in both hospitalized patients and ambulatory persons, can be classified into nine lipopolysaccharide (LPS) O-antigen serotypes. The O-antigen type has important clinical and epidemiological significance. However, K. pneumoniae O serotyping is cumbersome, and the reagents are not commercially available. To overcome the limitations of conventional serotyping methods, we aimed to create a rapid and accurate PCR method for K. pneumoniae O genotyping. We sequenced the genetic determinants of LPS O antigen from serotypes O1, O2a, O2ac, O3, O4, O5, O8, O9, and O12. We established a two-step genotyping scheme, based on the two genomic regions associated with O-antigen biosynthesis. The first set of PCR primers, which detects alleles at the wzm-wzt loci of the wb gene cluster, distinguishes between O1/O2, O3, O4, O5, O8, O9, and O12. The second set of PCR primers, which detects alleles at the wbbY region, further differentiates between O1, O2a, and O2ac. We verified the specificity of O genotyping against the O-serotype reference strains. We then tested the sensitivity and specificity of O genotyping in K. pneumoniae, using the 56 K-serotype reference strains with known O serotypes determined by an inhibition enzyme-linked immunosorbent assay (iELISA). There is a very good correlation between the O genotypes and classical O serotypes. Three discrepancies were observed and resolved by nucleotide sequencing-all in favor of O genotyping. The PCR-based O genotyping, which can be easily performed in clinical and research microbiology laboratories, is a rapid and accurate method for determining the LPS O-antigen types of K. pneumoniae isolates. Klebsiella pneumoniae is an encapsulated Gram-negative bacillus that frequently causes outbreaks of nosocomial infections in hospitalized patients (1-3). It can also affect ambulatory persons and cause community-acquired invasive diseases, including pyogenic liver abscess, endophthalmitis, meningitis, empyema, lung abscess, and necrotizing fasciitis (4-9). The worldwide emergence of multidrug-resistant strains and hypervirulent strains of K. pneumoniae has become an increasing clinical challenge and public health concern (10, 11).K. pneumoniae strains can be distinguished by their capsular polysaccharide (CPS) K-antigen types (77 serotypes) and lipopolysaccharide (LPS) O-antigen types (9 serotypes) (2). The K type and O type of K. pneumoniae both have important clinical and epidemiological significance (8, 12, 13). K1 clonal complex 23 is a newly emerged hypervirulent clade, which causes pyogenic liver abscesses with septic ocular or central nervous system complications (8,14,15). Likewise, O1 is associated with hypervirulent strains that cause pyogenic liver abscess (12). The O:K typing also helps establish the clonality of K. pneumoniae in nosocomial outbreak investigations (13). K-genotyping methods are now available for quickly and correctly determining the K types in K. pneumoniae (8,16,17). O...