Leaf trichomes are a very important morphological feature of tobacco plants and the metabolisms in these cells are associated with leaf quality and plant defense against pathogens and insects. However, effects of growth ecology regions on the gene expression in tobacco leaf trichomes have not been investigated in China. In this report, we characterize gene expression profiles in leaf trichomes of the most commonly commercial tobacco cultivar K326 (Nicotiana tabacum L cv. K326) grown in two main growth areas in China: Pingdingshan county in Henan Province, which is famous for producing strong aroma style leaf tobacco, and Yuxi County in Yunnan Province, which is famous for producing mild aroma style leaf tobacco. Tobacco plants were cultivated according to the farming practice implemented routinely in the two different localities in 2009. To characterize gene expression of leaf trichomes of tobacco growing in different ecological regions, cDNA microarray analysis was performed between trichomes of the same variety. Trichomes were collected from leaves through frozen brush way at the same position (9-11th), at the same development stage (60 d). Total RNA samples were isolated from trichomes and treated with DNAase to remove potential contamination of genomic DNA. The first strand cDNA was synthesized with a reverse-transcription. A 2831-EST database was established by trichome cDNA library sequencing in previous research. The 2831 trichome cDNAs were amplified by PCR technology and spotted on glass microscope slides to obtain cDNA chips for hybridization and microarray analysis. Ttrichome RNA samples from two different areas were prepared for labeling of the cDNA (Cy3 versus Cy5). In the first experiment, samples from Henan was labeled with Cy3 and samples from Yunnan was labeled with Cy5, and in the second experiment, labeling was reversed. Hybridization and microarray analysis were completed by following the protocol provided by Shanghai Boxing Gene Chips, Inc. The results showed that 445 genes were differentially expressed (with ratio values ≥2 or ≤0.5)between samples in the two areas. We observed that among these genes, the expression levels of 205 ones in samples from Henan Province were higher than in those from Yunnan Province. On the other hand, the expression levels of 240 genes in samples from Yunnan Province were higher than in those from Hunan Province. Functional analysis and gene clustering of those differentially expressed genes were molecularly characterized. The resulting data showed that genes involved in photosynthesis, carbohydrate biosynthesis, and terpenoid biosynthesis were expressed at higher levels in samples from Henan Province than in samples from Yunnan Province. In contrast, certain genes involved in carbohydrate and protein catabolism were expressed at higher levels in samples from in Yunnan Province than in those from Henan Province. Besides, these data showed that certain genes involved in resistance and defense were differentially expressed in samples from the two regions, likely indicating their different responses to two specific growth environmental circumstances. These observations imply that these differential gene expression profiles are likely associated with leaf aroma and thus are of significance in tobacco agriculture.