Armillaria species are important root rot pathogens with a wide host range and a worldwide distribution. The taxonomy of these fungi has been problematic for many years but the understanding of the relationships between them has been substantially improved through the application of DNA sequence comparisons. In this study, relationships between different Armillaria species were determined using elongation factor 1-alpha DNA sequence data for the first time. A total of 42 isolates, representing the majority of Armillaria species, with diverse geographic distributions and hosts, were included in this study. PCR amplification yielded products of 600 bp for all the isolates. Phylogenetic trees resulting from parsimony analysis showed that this gene region is useful for studying relationships between species. Generally, results were similar to those emerging from previous comparisons using ITS and IGS-1 sequence data. Phylogenetic trees generated from the dataset grouped the African taxa in a strongly supported clade, basal to the rest of the Armillaria species included in the study. The Armillaria species originating from the Northern Hemisphere formed a monophyletic group. Within this group, isolates of A. mellea constituted four subclades, representing their geographical origin. The phylogenetic relationships among species from the Southern Hemisphere were not entirely resolved. However, A. pallidula, A. fumosa and A. hinnulea grouped in a strongly supported clade and isolates of A. limonea formed a sister clade with those of A. luteobubalina. This is the first time a single-copy protein coding gene has been used to study phylogenetic relationships in Armillaria, and overall the data support previously held views regarding the relationships between species.