MicroRNAs (miRNAs) are tiny (20-24 nt bp) regulatory non-protein-coding RNA molecules that have been extensively characterized and found important for many physiological and developmental processes. The miss-expression of miRNAs leads to various defects in plants. MicroRNAs repress gene expression by directing mRNA degradation or translational arrest. Several proteins such as PP43A, HYL1, DCL, HST are indispensable role players in promoting miRNA biogenesis in plants. During miRNA biogenesis, lariat RNAs are produced as by-products of pre-mRNA splicing which have a negative role in regulation of miRNA homeostasis. By acting as a decoy and by sequestering to the dicing complex, lariat RNA can prevent the processing of miRNAs. A number of bioinformatic tools with different methodologies are available to identify and validate miRNAs and their targets. Many miRNAs have been reported in different crops for different traits; however, no reports are available on their use in plant breeding. Recently, researchers have developed trait specific miRNA-based molecular markers (miRNA-SSRs/SNP) for many quantitative traits in different plant species. In the future, these molecular markers can be used for plant breeding programs. In this review, a comprehensive up-to-date information is provided on the bioinformatic tools used for analysis of plant miRNAs and their targets, the number of miRNAs, their biogenesis, gene silencing mechanism and miRNA-based molecular markers.