MicroRNA (miRNA) is one class of newly identified, small, non-coding RNAs that play versatile and important roles in post-transcriptional gene regulation. All miRNAs have similar secondary hairpin structures; many of these are evolutionarily conserved. This suggests a powerful approach to predict the existence of new miRNA orthologs or homologs in other species. We developed a comprehensive strategy to identify new miRNA homologs by mining the repository of available ESTs. A total of 481 miRNAs, belonging to 37 miRNA families in 71 different plant species, were identified from more than 6 million EST sequences in plants. The potential targets of the EST-predicted miRNAs were also elucidated from the EST and protein databases, providing additional evidence for the real existence of these miRNAs in the given plant species. Some plant miRNAs were physically clustered together, suggesting that these miRNAs have similar gene expression patterns and are transcribed together as a polycistron, as observed among animal miRNAs. The uracil nucleotide is dominant in the first position of 5′ mature miRNAs. Our results indicate that many miRNA families are evolutionarily conserved across all major lineages of plants, including mosses, gymnosperms, monocots and eudicots. Additionally, the number of miRNAs discovered was directly related to the number of available ESTs and not to evolutionary relatedness to Arabidopsis thaliana, indicating that miRNAs are conserved and little phylogenetic signal exists in the presence or absence of these miRNAs. Regulation of gene expression by miRNAs appears to have existed at the earliest stages of plant evolution and has been tightly constrained (functionally) for more than 425 million years.