Eucalyptus is one of the world's main sources of biomass. The genus includes species representing the principle hardwood trees used for pulp and paper. Here, we aimed to identify genes specifically expressed in differentiating secondary xylem compared with phloem. We constructed a xylem vs phloem subtractive library (Xp) that generated 263 unique sequences. By transcript profiling of xylem, phloem, vascular cambium and leaves using macroarrays, we classified the 263 unigenes into distinct tissue-specific groups. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the differential expression of representative expressed sequence tags (ESTs). A total of 87 unigenes were preferentially expressed in xylem. They were involved in functional categories known to play roles in xylogenesis, such as hormone signaling and metabolism, secondary cell wall thickening and proteolysis. Some of these genes, including unknown genes, may be considered xylem-specific and they are likely to control important functions in xylogenesis. These data shed light on the cellular functions of xylem cells and, importantly, provide us with a portfolio of Eucalyptus xylem genes that may be major players in the control of wood formation and quality.