Technical Abstract: We describe a multiplex polymerase chain reaction assay (mPCR) for the identification and discrimination between isolates of Campylobacter coli, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. The Campylobacter jejuni isolate F38011 lpxA gene was identified by sequence analysis of an expression plasmid that restored wild-type lipopolysaccharide levels in Escherichia coli strain SM105 (lpxA). Using oligonucleotide primers developed to the C. jejuni lpxA gene, nearly full-length lpxA amplicons were amplified by the polymerase chain reaction from an additional 13 isolates of C. jejuni, 20 isolates of C. coli, 16 isolates of C. lari and five isolates C. upsaliensis. The nucleotide sequence for each amplicon was determined and alignment of the sequences, beginning at position -3 base pairs (relative to the C. jejuni lpxA gene) through to +745 base pairs, revealed a high level of species discrimination. Oligonucleotide primers were developed to exploit species differences and a mPCR assay was developed to positively identify isolates of C. coli, C. jejuni, C. lari and C. upsaliensis. We characterized an additional set of thermotolerant isolates by nucleotide sequence analysis to further demonstrate the uniqueness of each species-specific region. The mPCR assay was validated using 112 genetically-defined isolates of C. coli, C. jejuni, C. lari and C. upsaliensis, 35 strains representing twelve additional Campylobacter species, and 25 strains representing 20 non-Campylobacter species. Application of the mPCR method to whole cell lysates obtained from 108 clinical and environmental thermotolerant Campylobacter isolates resulted in 100% correlation with other typing methods.