Most species of Ceratocystis sensu stricto are virulent pathogens of a wide variety of plants including forest and fruit trees, sweet potato, pineapple and sugar cane. Confusion exists regarding the taxonomy of the species in this genus. The aim of this study was to develop a rapid and reliable PCR-based RFLP identification method and to consider phylogenetic relationships among the better-known species of Ceratocystis. A 1·6 kb fragment within the ribosomal DNA operon was directly amplified from living fungal tissue, without extracting DNA. The amplified fragment included part of the small (SSU) and large (LSU) sub-unit rRNA genes, the 5·8S rRNA gene and the internal transcribed spacers (ITS) 1 and 2. The PCR fragments were digested with eighteen restriction enzymes. Four of these (Alu I, Dra I, Hae III and Rsa I) produced RFLPs that separated the species of Ceratocystis. The amplification products from the best-known species were sequenced, and the delimitation of taxa based on this phylogenetic analysis generally agreed with results of previous studies using isozymes and rDNA sequence analysis. This study provides an extended understanding of the relationships among species of Ceratocystis and will form a sound foundation for further taxonomic studies of the group.