Abstract Background Transcriptomics has resulted in the development of large data sets and tools for the progression of functional genomics and systems biology in many model organisms. Currently there is no commercially available microarray to allow such expression studies in Nicotiana tabacum (tobacco). Results A custom designed Affymetrix tobacco expression microarray was generated from a set of over 40k unigenes and used to measure gene expression in 19 different tobacco samples to produce the Tobacco Expression Atlas (TobEA). TobEA provides a snap shot of the transcriptional activity for thousands of tobacco genes in different tissues throughout the lifecycle of the plant and enables the identification of the biological processes occurring in these different tissues. 772 of 2513 transcription factors previously identified in tobacco were mapped to the array, with 87% of them being expressed in at least one tissue in the atlas. Putative transcriptional networks were identified based on the co-expression of these transcription factors. Several interactions in a floral identity transcription factor network were consistent with previous results from other plant species. To broaden access and maximise the benefit of TobEA a set of tools were developed to provide researchers with expression information on their genes of interest via the Solanaceae Genomics Network (SGN) web site. The array has also been made available for public use via the Nottingham Arabidopsis Stock Centre microarray service. Conclusions The generation of a tobacco expression microarray is an important development for research in this model plant. The data provided by TobEA represents a valuable resource for plant functional genomics and systems biology research and can be used to identify gene targets for both fundamental and applied scientific applications in tobacco.