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Background: As a large class of endogenous and small non-coding RNAs, miRNAs (miRNAs) play fundamental roles in multiple biological processes.With the development of the deep sequencing technology, more and more novel miRNAs detected in organisms, has been greatly accelerated the research of miRNAs.Specially, the silkworm, Bombyx mori, an important economic insect, is not only used for silk production, but also widely employed to investigate fundamental biological phenomena as a powerful model organism.With an aim to expanding our knowledge on the role of miRNAs played in silk synthesis, we sequenced six small RNA libraries prepared from posterior silk glands in four strains of silkworm and two development stages, specifically, 4th-instar molting to 5th-instar day-2 and 5th-instar day-3 to 5th-instar day-8 before spinning.Methods: We employed large-scale Solexa sequencing technology to extend the repertoire of miRNAs in silkworm posterior silk glands.With the aid of deep sequencing small RNA analysis pipeline (DSAP) and MIREAP, the known and candidate novel miRNAs were obtained for further analysis.For identifying the real miRNA precursor from the pseudo ones, three SVM-based prediction programs, Triplet-SVM, PMirp and Mirident, based on training models of known human miRNA precursors, were selected for comparison the performance in identifying known miRNA precursors of 24 species of insects.We further integrated known miRNAs from several kinds of fly and the entire silkworm known miRNAs in miRBase 18.0 as well as other novel silkworm miRNAs identified from latest published articles as probes for microarray identification.Subsequently, Bioinformatics analysis such as TargetScan, gene ontology and KEGG pathway analyses were used for further analysis of the target gene of the differentially expressed miRNAs.Results: Totally, 574 novel miRNAs in which 52 miRNA/miRNA* duplex were identified from solexa sequencing.There were 382 mature miRNAs confirmed by microarray and might express in 5th-instar day-3, and among them, 68 miRNAs were proved to be novel miRNAs including 19 conserved in Drosophila.Bioinformatics analysis of the novel miRNA targets further illustrated the likely role for differentially expressed miRNAs in posterior silk glands.Conclusions: The newly identified miRNAs largely enrich the repertoire of silkworm miRNAs.Moreover, comparative analysis of known and novel miRNAs indicated that the expression level of miRNAs might be the key reason to interpret different yields of cocoon .