Technical Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression in eukaryotes modulating a large diversity of biological processes. The discovery of miRNAs has provided new opportunities to understand the biology of a number of species. The cattle tick Rhipicephalus (Boophilus) microplus causes significant economical losses in cattle production worldwide and the lack of an effective vaccine requires that several approaches be conducted to further understand their biology. To be able to provide new insights into the biology of cattle ticks and to expand the repertoire of tick miRNAs we utilized Illumina/Solexa technology to sequence the small RNA transcriptomes derived from various tick life cycles stages and selected organs. To discover and profile novel tick miRNAs we employed a comparative genomic approach identifying identical or nearly identical miRNAs conserved between cattle ticks and flies. Overall we found 58 tick miRNAs with 33, 49 and 48 of these expressed in eggs, larvae and adult ticks, respectively. We also found 42, 41 and 38 miRNAs expressed in the female tick gut, salivary glands and ovaries, respectively. The major differences in miRNA among samples results from differential expression levels of commonly expressed miRNAs. The only previously known tick miRNA, miR-9a, was found expressed in all samples sequenced in this study, but particularly up regulated in tick larvae exposed for six hours to their host. The most abundant tick miRNA found in this study is miR-1 particularly highly expressed in larval and adult stages as well as female gut and ovaries. In salivary glands we found a significant up regulation of let-7 and miR-275 making these miRNAs interesting targets for further functional studies. Findings presented in this study extend our knowledge of the biology and evolution of microRNAs in arthropods. The repertoire of tick microRNAs has been significantly expanded in this study and we envisage that these resources will facilitate functional annotation of the cattle tick genome and promote further functional and comparative genomics studies of small regulatory RNAs in arthropods.