Campylobacter hyointestinalis is considered as an emerging zoonotic pathogen. We have recently identified two types of cytolethal distending toxin (cdt) gene in C. hyointestinalis and designated them as Chcdt-I and Chcdt-II. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay that can differentiate Chcdt-I from Chcdt-II. When the PCR-RFLP assay was applied to 17 other Campylobacter strains and 25 non-Campylobacter strains, PCR products were not obtained irrespective of their cdt gene-possession, indicating that the specificity of the PCR-RFLP assay was 100%. In contrast, when the PCR-RFLP assay was applied to 35 C. hyointestinalis strains including 23 analyzed in the previous study and 12 newly isolated from pigs and bovines, all of them showed the presence of cdt genes. Furthermore, a restriction digest by EcoT14-I revealed that 29 strains contained both Chcdt-I and Chcdt-II and 6 strains contained only Chcdt-II, showing 100% sensitivity. Unexpectedly, however, PCR products obtained from 7 C. hyointestinalis strains were not completely digested by EcoT14-I. Nucleotide sequence analysis revealed that the undigested PCR product was homologous to cdtB but not to Chcdt-IB or Chcdt-IIB, indicating the presence of another cdt gene-variant. Then, we further digested the PCR products with DdeI in addition to EcoT14-I, showing that all three cdt genes, including a possible new Chcdt variant, could be clearly differentiated. Thus, the PCR-RFLP assay developed in this study is a valuable tool for evaluating the Chcdt gene-profile of bacteria.