MicroRNAs (miRNAs) are a new family of small RNA molecules found in plants and animals. We developed a comprehensive strategy for identifying new miRNA homologues by mining the repository of available citrus expressed sequence tags (ESTs). By adopting a range of filtering criteria, we identified a total of 38 potential miRNAs - nine, five, nine and 15 miRNAs in Citrus trifoliata (ctr-miRNAs), C. clementina (ccl-miRNAs), C. reticulata (crt-miRNAs) and C. sinensis (csi-miRNAs), respectively - from more than 430,000 EST sequences in citrus. Using the potential miRNA sequences, we conducted a further BLAST search of the mRNA database and found six potential target genes in these citrus species. Eight miRNAs were selected in order to verify their existence in citrus using Northern blotting, and the functions of several miRNAs in miRNA-mediated gene regulation are experimentally suggested. It appears that all these miRNAs regulate expression of their target genes by cleavage, which is the most common situation in gene regulation mediated by plant miRNAs. Our achievement in identifying new miRNAs in citrus provides a powerful incentive for further studies on the important roles of these miRNAs.