The cattle tick, Rhipicephalus (Boophilus) microplus, causes significant economic losses in cattle production worldwide. To provide new insights into the biology of cattle ticks we utilized a miR-Seq approach to sequence small RNAs derived from various life stages and selected organs. We found 51 evolutionary conserved R. microplus miRNA loci, with 36 of these previously found in the tick Ixodes scapularis. The majority of the R. microplus miRNAs are perfectly conserved throughout evolution with 11, 5 and 15 of these conserved since the Nephrozoan (640 MYA), Protostomian (620MYA) and Arthropoda (540 MYA) ancestor, respectively. We then employed a de novo computational screening for novel tick miRNAs using the draft genome of I. scapularis and genomic contigs of R. microplus as templates. This identified 36 novel R. microplus miRNA loci of which 12 were conserved in I. scapularis. Overall we found 87 R. microplus miRNA loci, of these 15 showed the expression of both miRNA and miRNA* sequences. R. microplus miRNAs showed a variety of expression profiles, with the evolutionary-conserved miRNAs mainly expressed in all life stages at various levels, while the expression of novel tick-specific miRNAs was mostly limited to particular life stages and/or tick organs. Interestingly, anciently acquired miRNAs in the R. microplus lineage tend to accumulate the least amount of nucleotide substitutions as compared to more recently acquired miRNAs. This correlates with the notion that anciently acquired miRNAs may play key cellular functions in a range of species and are under selective pressure to remain unchanged throughout evolution.