ABSTRACT We describe a multiplex PCR assay to identify and discriminate between isolates of Campylobacter coli , Campylobacter jejuni , Campylobacter lari , and Campylobacter upsaliensis . The C. jejuni isolate F38011 lpxA gene, encoding a UDP- N -acetylglucosamine acyltransferase, was identified by sequence analysis of an expression plasmid that restored wild-type lipopolysaccharide levels in Escherichia coli strain SM105 [ lpxA (Ts)]. With oligonucleotide primers developed to the C. jejuni lpxA gene, nearly full-length lpxA amplicons were amplified from an additional 11 isolates of C. jejuni , 20 isolates of C. coli , 16 isolates of C. lari , and five isolates of C. upsaliensis . The nucleotide sequence of each amplicon was determined, and sequence alignment revealed a high level of species discrimination. Oligonucleotide primers were constructed to exploit species differences, and a multiplex PCR assay was developed to positively identify isolates of C. coli , C. jejuni , C. lari , and C. upsaliensis. We characterized an additional set of 41 thermotolerant isolates by partial nucleotide sequence analysis to further demonstrate the uniqueness of each species-specific region. The multiplex PCR assay was validated with 105 genetically defined isolates of C. coli , C. jejuni , C. lari , and C. upsaliensis , 34 strains representing 12 additional Campylobacter species, and 24 strains representing 19 non- Campylobacter species. Application of the multiplex PCR method to whole-cell lysates obtained from 108 clinical and environmental thermotolerant Campylobacter isolates resulted in 100% correlation with biochemical typing methods.