Polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) analysis of ribosomal (r) DNA was conducted on Uncinaria stenocephala, Ancylostoma caninum, A. tubaeforme and A. ceylanicum. The rDNA region spanning the first and second internal transcribed spacers (ITS1 and ITS2) plus the 5.8S (ITS+) gene was amplified by PCR from each of the species, digested separately with 9 restriction endonucleases and the fragments separated by agarose gel electrophoresis. Characteristic PCR-RFLP patterns were produced for each morphologically defined species using some of the endonucleases. The present study demonstrated that the ITS+ provides genetic markers for the delineation of each species examined and suggests that this region of rDNA will be useful for the identification of other hookworms from a range of hosts. The results are likely to have important implications for studying the genetic structure of hookworm populations, the systematics and the epidemiology of hookworm infections.