Abstract: MicroRNAs (miRNAs) are endogenous non-protein-coding RNAs of approximately 22 nucleotides. Thousands of miRNA genes have been identified (computationally and/or experimentally) in a variety of organisms, which suggests that miRNA genes have been widely shared and distributed among species. Our laboratory has applied mature miRNA sequence patterns to scan the genome sequences of 56 bilaterian animal species for identification of candidate miRNAs. The regions centered surrounding these candidate miRNAs were then extracted for folding and calculating the features of their secondary structure. Using a support vector machine as a classifier combined with these features, we identified more than 15,000 orthologous or paralogous candidate pre-miRNAs, as well as their corresponding candidate mature miRNAs. Stem-loop RT-PCR and deep sequencing methods were used to experimentally validate the prediction results in human, medaka and rabbit. In addition, we have identified 38 methylation-associated miRNAs in human gastric cancer cells. The methylation-silenced expression of these microRNAs could be reactivated in gastric cancer cells by treatment with demethylating drugs in time-dependent manner. Analysis of the methylation status of these miRNAs showed that the upstream CpG-rich regions of several miRNAs are frequently methylated in gastric cancer tissues compared with adjacent normal tissues, and their methylation status correlated inversely with their expression patterns. These miRNAs silenced by tumor-specific methylation could play significant oncogenesis roles in gastric cancer progression.