For studies on functional genomics, small RNAs, especially microRNAs (miRNAs), have emerged as a hot topic due to their importance in cellular and developmental processes. Identification of insect miRNAs largely depends on the availability of genomic sequences in the public domain. The large milkweed bug, Oncopeltus fasciatus (Dallas) is a hemimetabolous insect which has become a model hemipteran system for various molecular studies. In this study, we identified 96 candidate mature miRNAs from O. fasciatus genome using a blast search with the previously reported animal miRNAs. The secondary structure of predicted miRNA sequences was determined online using "mfold" web server and verified by calculating the minimal free energy index (MFEI). Six miRNAs let-7e, miR-133c, miR-219b, mir-466d, mir-669f, and mir-669l are reported for the first time in Insecta. Comparison of O. fasciatus mir-2 and mir-71 family clusters to those of diverse insect species showed that they are highly conserved. The phylogenetic analysis of miRNAs revealed the evolutionary relationship of conserved miRNAs of O. fasciatus with other insect species. Using a classical rule-based algorithm method, we predicted the possible targets of the new miRNAs. Our study not only identified the list of miRNAs in O. fasciatus but also provides a basic platform for developing novel pest management strategies based on artificial miRNAs.