Temporal and spatial regulation of gene expression plays a critical role in shaping tobacco growth and development. Many genes show considerable variation in expression between different organs and developmental stages in other plant species and changes in expression even occurs on a daily basis for genes involved in various aspects of plant physiology and metabolism. Understanding where and when expression occurs will give us valuable insight into gene function and the functioning of the plant as a whole. Using a custom designed AffymetrixTM micro-array, the expression of 43, 723 Nicotiana tabacum (var. K326) genes was measured in a range of different organs and developmental stages. Gene expression in leaves was also measured in a time-course during two days of diurnal conditions. Principal Component Analysis of gene expression differences allowed distinction between samples from different tissues. Consistent with results from other plant species, there was a clear distinction between tissues from the root or shoot. The Tobacco Expression Atlas (TobEA) provides a valuable resource for both functional genomics and systems biology research in tobacco.