The aim of the present work was to determine the identity and molecular relationships between 127 strains of dark septate (DS) fungi isolated from healthy root tips, decayed coarse roots, live healthy-looking stems, coarse (stumps, snags and logs) and fine (tree branches and tops) woody debris in temperate-boreal forests in Sweden and Lithuania. Sequence analysis of ITS rDNA was used to identify the fungi. In a neighbour-joining similarity tree, all sequences were grouped into five distinct clusters. Within each of these, ITS rDNA sequence variation consisted of 2–18 nucleotides, corresponding to 1–3% of their total length. The four least variable clusters were supported with high bootstrap values of 86–100%. Comparisons with the sequences in the GenBank database showed that all our strains had a 95–100% homology with identified Phialocephala species, and they were thus assigned to this genus. The representatives of two clusters were identified, as P. fortinii and P. dimorphospora. The representatives of three remaining clusters were defined as Phialocephala sp. 35, Phialocephala sp. 6 and Phialocephala sp. 18. Within each of these clusters, ITS rDNA sequence uniformity was higher than that observed within P. fortinii and P. dimorphospora. Consequently, their clusters were most discrete, supported with bootstrap values of 100%. Genetic variation in the five distinguished Phialocephala species and their possible ecological roles are discussed. Phialocephala sp. 6 was confined to healthy root tips of conifers. P. dimorphospora was only associated with dead woody tissue of P. abies. P. fortinii, Phialocephala sp. 18 and sp. 35 were isolated from both dead and living conifers and Betula pendula. In conclusion, the present study revealed the ability of fungi from the genus Phialocephala to colonise and persist in live and dead trees under strikingly different ecological conditions.