Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes.