MicroRNAs (miRNAs) are recently discovered short regulatory RNA molecules representing a new layer in posttranscriptional gene expression regulation. Although more than 450 human miRNAs have been identified, only a very few of them have been characterized in detail. The precise understanding of miRNA-mediated processes requires the reliable spatial and temporal analyses of miRNA accumulation at tissue/cell level. However, the detection of miRNAs by in situ hybridization (ISH) is technically challenging because of the small size of target sequences. It was shown recently that locked nucleic acid nucleotide-containing probes can anneal to short nucleic acids with high specificity. This enabled several research groups to analyze the expression patterns of miRNAs in both plant and animal tissues. This review focuses on the results of recent publications on the detection of miRNAs by ISH.