Suppression subtractive hybridization (SSH) is an effective approach to identify the genes that vary in expression levels during different biological processes. It is often used in higher eukaryotes to study the molecular regulation in complex pathogenic progress, such as tumorigenesis and other chronic multigene-associated diseases. Because microbes have relatively smaller genomes compared with eukaryotes, aside from the analysis at the mRNA level, SSH as well as its modifications have been further employed to isolate specific chromosomal locus, study genomic diversity related with exceptional bacterial secondary metabolisms or genes with special microbial function. This review introduces the SSH and its associated methods and focus on their applications to detect specific functional genes or DNA markers in microorganisms.