The internal transcribed spacers, ITS1 and ITS2, and 5.8S region of ribosomal DNA (rDNA) from 20 species of Ascosphaera were amplified by PCR, and their sequences determined and compared. No variation was detected in the sequences from 23 widely distributed isolates of A. apis, in sequences from 11 widely distributed A. atra isolates, in four A. aggregata isolates, or in sequences from two isolates each of A. acerosa, A. duoformis, A. flava, A. larvis, A. pollenicola and A. proliperda. However, the ribosomal sequences from each of these nine species, and from another 11 species of which only a single isolate was examined, differed from one another by 0·18–30·9%. Thus these sequences provide a rapid method for identifying species, and searches using them showed that the sequence of A. apis rDNA recorded in the international databases is, in fact, that of A. atra.

The rDNA sequences also provided data for assessing the relationships of these fungi. Of the rDNA sequences in current international databases, that of Eremascus albus was very close to, but distinct from, those of the Ascosphaera species. Comparisons of the Ascosphaera sequences showed that most formed consistent clusters irrespective of the method of comparison used (distance matrix and parsimony), or whether the ITS1 or ITS2 portions were used; A. acerosa with A. asterophora, A. atra with A. duoformis, A. colubrina closely with A. flava, A. larvis, A. major, A. variegata and more distantly with A. apis and A. celerrima, and, also, A. aggregata with A. subcuticulata, A. proliperda and more distantly with A. solina. The apparent relationships of these clusters were inconsistent, depended on the alignment of several regions of ‘indels’, and could not be resolved. The A. fusiformis, A. naganensis and A. osmophila sequences showed inconsistent relationships with the others, especially that of A. osmophila, which had an A. solina-like ITS2 region, but an atypical ITS1 sequence with a large unique repetitive insertion. The clusters based on gene sequence comparisons clearly correlated with groupings based on ascospore morphology and other characters.