Perennial woody plants differ from annual herbaceous plants in several ways and are expected to have evolved to adopt a unique repertoire and expression profiles of functional genes. Poplar, a model tree species for which a large number of ESTs are publicly available, was used to carry out a large-scale comparative analysis with the expressed sequences of eight plant species. First, we obtained 105,831 poplar ESTs from public databases and identified a set of 25,282 unigenes (i.e., tentative non-redundant sequences). The majority of the unigenes (56%) had significant matches to Arabidopsis genes. We then estimated poplar multigene families by counting the tBLASTX matches of each unigene against the poplar unigene dataset itself. Forty-seven percent of the 25,282 unigenes were subsequently organized into 3,481 multigene families 89% of which had less than five copy members. In poplar, protein kinases represent the largest family followed by GTP-binding proteins and Myb transcription factors. Several multigene families had a higher copy number in poplar than in Arabidopsis hinting potential lineage-specific proliferation of poplar protein families. Such expansion may be related to the adaptation of perennial poplars for the high degree of environmental stresses that affects growth and survival. Comparison of poplar unigenes with the Arabidopsis transcriptome revealed that genes involved in transcriptional regulation are the most divergent while metabolism-related genes are the most conserved.