Full-length coding sequences of the β-tubulin gene (tubA) were PCR-amplified and sequenced from 42 Phaeosphaeria isolates, including 16 P. nodorum and 23 P. avenaria species from cereals, two Polish isolates from rye (Secale cereale L.), and one isolate from dallis grass (Paspalum dilatatum Poir). A tubA gene of size 1556 bp was identified in wheat- and barley-biotype P. nodorum (PN-w and PN-b), P. avenaria f. sp. avenaria (Paa), homothallic P. avenaria f. sp. triticea (Pat.) (Pat1) and the Pat. isolate (Pat3) from the State of Washington. The tubA gene length polymorphisms were detected in two Pat. isolates (Pat2) from foxtail barley (Hordeum jubatum L.), one from dallis grass and two Polish isolates from rye. These size differences were due to the variation of intron lengths among these three Phaeosphaeria species. All Phaeosphaeria isolates have identical 1344 bp exons that can be translated into a 447 amino acid β-tubulin. Like glyceraldehyde-3-phosphate dehydrogenase, the β-tubulin amino acid sequence was identical in all Phaeosphaeria species used in this study, with the exception of the two Pat2 isolates. Six amino acid differences were evident in the β-tubulin of these Pat2 isolates.