A multilocus sequence typing (MLST) system has been reported previously for Campylobacter jejuni to both differentiate strains and identify clonal lineages. However, sequence variation at the MLST loci prevents its use for closely related Campylobacter species. We describe herein an expanded MLST method to include three clinically relevant Campylobacter species, C. coli, C. lari, and C. upsaliensis, and a fourth Campylobacter species, C. helveticus. The C. coli and C. helveticus methods use the same seven C. jejuni loci (aspA, atpA, glnA, gltA, glyA, pgm, and tkt); however, adk and pgi were substituted for aspA and gltA in C. lari and for gltA and pgm in C. upsaliensis. Multiple C. coli (n = 57), C. lari (n = 20), C. upsaliensis (n = 78), and C. helveticus (n = 9) isolates, representing both clinical and environmental sources, were typed. All four species were genetically diverse: the majority (> 80%) of the isolates had unique sequence types (STs). Using this method, mixed C. lari, C. upsaliensis, and C. helveticus isolates were identified; upon separation, each isolate was shown to contain two strains of the same species with distinct STs. Additionally, the expanded MLST method was able to detect potential lateral transfer events between C. jejuni and either C. coli or C. lari and between C. upsaliensis and C. helveticus. Thus, the expanded MLST method will prove useful in differentiating strains of five Campylobacter species, identifying mixed Campylobacter cultures, and detecting genetic exchange within the genus.