MicroRNAs (miRNAs) are small non-coding RNAs that regulate various aspects of gene expression in physiology and development. Links between miRNAs and the initiation and progression of human diseases are becoming increasingly apparent. The development of methods to detect the subcellular and tissue localization of miRNAs is essential for understanding their biological role in homeostasis. In this review, we discuss how in situ hybridization can complement tissuelevel miRNA expression profiling and its role as an investigational tool to better understand the etiology of human diseases. Furthermore, in situ hybridization of miRNAs represents a potent diagnostic assay that could be further refined and utilized for clinical applications.