Restriction enzyme analysis of ribosomal DNA (rDNA) sequences was used to distinguish species and isolates of root-knot nematodes. DNA fragments containing the internal transcribed spacer (ITS) rDNA were amplified from total DNA of 27 geographic isolates each of Meloidogyne hapla and M. chitwoodi and from one isolate each of M. incognita and M. javanica by polymerase chain reaction (PCR). The amount of DNA present in a single juvenile was sufficient to amplify these PCR products. The amplified ITS fragments were relatively short compared to those that have been found for the genera Aphelenchoides, Caenorhabditis, Ditylenchus, Heterodera, and Xiphinema. Digestion of the ITS regions with AluI, DraI, and HinfI distinguished M. hapla and M. chitwoodi from each other as well as from M. incognita and M. javanica. Results indicated that different ITS sequences are present within a single individual of M. hapla. Three isolates of M. chitwoodi that produce isozyme patterns distinct from other M. chitwoodi isolates also could be distinguished by ITS restriction fragment length polymorphisms. The possibility that they do not belong to M. chitwoodi proper is discussed.