Abstract.. Ceratocystis sensu stricto includes numerous species of insect-vectored, wood-staining and plant pathogenic fungi. Among these, Ceratocystis coerulescens is a well-known cause of blue-stain in spruce and pine. Previous investigations, using morphological characteristics and isozyme comparisons, have shown that C. coerulescens encompasses at least five morphological types. The aim of this study was thus to compare isolates of C. coerulescens sensu lato and morphologically similar species, including C. laricicola, C. polonica, C. virescens, C. eucalypti, Chalara australis and Ch. neocaledoniae, on the basis of DNA sequence data. Using the polymerase chain reaction (PCR), a 600 base pair fragment within the ribosomal DNA operon was amplified, and the PCR products were sequenced. The analyzed sequence included the 5.8S rRNA gene and the internal transcribed spacers (ITS) 1 and 2. Relationships were determined by parsimony analysis. Using C. fimbriata as the outgroup taxon, the five morphological types previously known as C. coerulescens and the two other taxa from conifers formed a strongly-supported monophyletic group that includes all the Ceratocystis species occurring primarily on conifers. The species from hardwood trees, C. eucalypti, Ch. australis and Ch. neocaledoniae, also formed a monophyletic group, sister to the conifer group. The fourth species from hardwoods, C. virescens, formed a group basal to the two sister groups.