Promoters are crucial regulatory sequences that enable a gene to be transcribed in specific organs or tissues. Recovery of promoter sequences, especially those leading to tissue-specific expression, is of great interest in plant biotechnology. In this study, an in silico analysis of the Brazilian Coffee Genome Project Database was accomplished to identify genes with organ-specific expression in coffee. Several ESTs showing root (30) and leaf-specific (14) expression patterns were selected, but only two of them, one in root and the other in leaf, were validated as being specific by RT-PCR experiments. The DNA regions immediately 5' to the validated ESTs were isolated by chromosome walking and amplified fragments of 2.0 (root) and 0.9 (leaf) kb in length were obtained. Using a web-base program, several cis-regulatory elements were identified within the amplified sequences, including those involved in the control of tissue-specificity and stress response. The isolated promoters were transcriptionally fused to the β-Glucuronidase (GUS) reporter gene and tested in transient expression assays using coffee seedlings. GUS histochemical analyses confirmed the functionality and expected organ-specificity of the cloned promoters. To our knowledge, these are the first regulatory sequences presenting root and leaf-specific expression cloned in coffee.