Diversity in host range, pathogenicity, phenotypic characteristics, repetitive extragenic palindromic polymerase chain reaction (rep-PCR) profiles, and sequence of the 16S-23S rDNA spacer region was examined among 44 Xanthomonas strains isolated from lettuce. Forty-two of the strains were divided into two groups, designated A and B. Seventy percent were Group A, and most of the remaining strains including a reference strain (LMG 938) were Group B. Group A strains induced both local and systemic symptoms, whereas Group B strains caused only distinct necrotic spots. Two strains, including the X. campestris pv. vitians type strain, were distinct from the Group A and B strains and were not pathogenic on lettuce. Analysis of fatty acid profiles, serotype, carbon substrate utilization patterns, and protein fingerprints confirmed this grouping. The Group A and B strains also formed two unique clusters (I and II) by rep-PCR profiling that corresponded to the two groups. Direct sequencing of a PCR-amplified DNA fragment (680 bp) from the 16S-23S rDNA spacer region of four representative strains, however, did not differentiate these groups. Serology and rep-PCR fingerprinting can be used to diagnose and identify X. campestris pv. vitians strains, while the other analyses evaluated are useful for strain characterization.