Cellulose is one of the most abundant biopolymers on earth and is an important commodity for industries such as the pulp and paper industry. Cellulose is deposited into the plant cell walls by a complex of membrane bound enzymes known as cellulose synthases. A number of cellulose synthase (CesA) genes, which encode for different cellulose synthase proteins, have been identified from plant species such as Eucalyptus, Populus and Arabidopsis. Mutant and expression profile analysis of the CesA genes indicated that a set of three CesA genes are associated with secondary cell wall formation, while a different set of CesA genes are associated with primary cell wall formation. The aim of this study was to investigate the transcriptional regulation of the different members of the CesA gene family in Eucalyptus. The promoter regions were comparatively analysed with the orthologous regions in Arabidopsis and Populus using bioinformatics tools to identify putative regulatory motifs that playa role in CesA genes regulation. Six Eucalyptus CesA gene promoters were isolated using genome walking. The Eucalyptus promoter regions and the orthologous promoter regions from Populus and Arabidopsis were analysed using TSSP (Transcriptional start site plant promoter prediction) and NNPP (Neural network promoter prediction) software packages. The software packages predicted the transcriptional start sites of the genes and the core regulatory elements such as the TATA-box and initiator elements. The in silico results were compared among species and it was found that the predicted transcriptional start sites and the core elements of the CesA gene promoters showed substantial structural conservation. The promoter regions were used in a comparative in silico analysis with the orthologous promoter regions from Arabidopsis and Populus to identify putative regulatory motifs. This is the first study in which the promoters of the CesA gene family are characterized in Arabidopsis, Populus and Eucalyptus. Three software packages (Weeder, POCO and MotifSampler) were used to analyse the promoter regions and identify over-represented motif sequences. A number of key stem-specific and xylem-specific motifs such as the AC-motif and G-box motif were identified as well as a number of novel motifs. Although all of the predicted motifs identified here will have to be functionally tested, the results of this study provide a good map for directed deletion studies and functional testing of the CesA promoters.