MicroRNAs (miRNAs) are endogenous non-coding genes that participate in post-transcription regulation by either degrading mRNA or blocking its translation. It is considered to be very important in regulating insect development and metamorphosis. We conducted a large-scale screening for miRNA genes in the silkworm Bombyx mori using sequence-by-synthesis (SBS) deep sequencing of mixed RNAs from egg, larval, pupal, and adult stages. Of 2,227,930 SBS tags, 1,144,485 ranged from 17 to 25 nt, corresponding to 256,604 unique tags. Among these non-redundant tags, 95,184 were matched to the silkworm genome. We identified 3,750 miRNA candidate genes using a computational pipeline combining RNAfold and TripletSVM algorithms. We confirmed 354 miRNA genes using miRNA microarrays and then performed expression profile analysis on these miRNAs for all developmental stages. While 106 miRNAs were expressed in all stages, 248 miRNAs were egg- and pupa-specific, suggesting that insect miRNAs play a significant role in embryogenesis and metamorphosis. We selected eight miRNAs for quantitative RT-PCR analysis; six of these were consistent with our microarray results. In addition, we searched for orthologous miRNA genes in mammals, a nematode, and other insects and found that most silkworm miRNAs are conserved in insects, whereas only a small number of silkworm miRNAs has orthologs in mammals and the nematode. These results suggest that there are many miRNAs unique to insects.