MicroRNAs (miRNAs) are a class of non-protein coding small RNAs that regulate expression of genes at post-transcriptional levels. Increasing evidence has shown that miRNAs play multiple roles in biological processes, including development, cell proliferation and apoptosis. Based on the conservation of miRNAs sequence, using a computational homology search based on genomic survey sequence analysis, a total of 16 novel miRNAs were identified and characteristics such as family and evolutionary conservation have been described. By using these newly identified miRNAs, the mRNA database of silkworm was blasted and 21 potential targets of miRNAs were detected. Most of these miRNA targeted genes were predicted to encode transcription factors. The semi-stem loop RT-PCR based assays were performed and found that silkworm miRNAs have diverse expression patterns during development.