The ability to profile bacterial gene expression has markedly advanced the capacity to understand the molecular mechanisms of pathogenesis, epidemiology, and therapeutics. This advance has been coupled with the development of techniques that enable investigators to identify bacterial specifically expressed genes and promise to open new avenues of functional genomics by allowing researchers to focus on the identified differentially expressed genes. During the past two decades, a number of approaches have been developed to investigate bacterial genes differentially expressed in response to the changing environment, particularly during interaction with their hosts. The most commonly used techniques include in vivo expression technology, signature-tagged mutagenesis, differential fluorescence induction, and cDNA microarrays, which fall into two broad classes: mutagenesis-based technologies and hybridization-based technologies. Selective capture of transcribed sequences, a recently emerging method, is a hybridization-based technique. This technique is powerful in analyzing differential gene expression of the bacteria, with the superb ability to investigate the bacterial species with unknown genomic information. Herein, we describe the application of this technique in a comparative study of the gene expression between two closely related bacteria induced or repressed under a variety of conditions.