Abstract: Species-specific primers for Contracaecum rudolphii A, Contracaecum rudolphii B and Contracaecum septentrionale, namely HFA(F), HFA(R), HFB(F) and HFS(F), were designed based on sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of ribosomal DNA. Under optimal amplification conditions, specific rDNA fragments of 323bp, 321bp and 108bp in length were amplified by PCR for C. rudolphii A, C. rudolphii B and C.septentrionale, respectively, while no fragment was amplified from gDNAs from other parasites used as controls. The lowest DNA concentration that the assay could amplify was 1.6, 21.8 and 0.27 ng/μL for C. rudolphii A, C. rudolphii B and C.septentrionale, respectively. These assays could be used for the accurate and rapid identification of members of the C. rudolphii complex, and for the differentiation between C.septentrionale and the C. rudolphii complex. These methods could provide tools for the diagnosis and epidemiological survey of infections these anisakids caused.