Severe epidemics of bacterial spot have been observed in central-west Brazil in fields of processing tomato. Several xanthomonads, Xanthomonas axonopodis pv. vesicatoria, X. vesicatoria, or X. gardneri, can cause the disease; therefore, attempts were made to identify the pathogen species present in this region. A total of 215 strains were obtained from 10 commercial areas in 1997, 1998, and 2000. The strains were characterized using pulsed-field gel electrophoresis (PFGE) and by their amylolytic and pectolytic activities. Representative strains from each PFGE haplotype then were tested for pathogenicity on tomato and pepper, carbon source utilization, and whole protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis. rRNA sequence comparisons also were performed. All strains recovered from six fields were classified as X. gardneri, whereas X. vesicatoria and X. axonopodis pv. vesicatoria also were detected in the remaining four fields. Strains of X. gardneri, which could be grouped into two PFGE haplotypes, were unable to hydrolyze starch and pectate and to utilize gentiobiose and maltose. They expressed the β protein of 27 kDa and were pathogenic on tomato but variable on pepper. This is the first report of outbreaks of bacterial spot on tomato caused by X. gardneri.