MicroRNAs are small ~22 nucleotide-long non-coding RNAs capable of controlling gene expression by inhibiting translation. Alignment of human microRNA stem loop sequences (mir) against a recent draft sequence assembly of the bovine genome resulted in identification of 334 predicted bovine mir. We sequenced five tissue-specific cDNA libraries derived from the small RNA fractions of bovine embryo, thymus, small intestine, and lymph node to validate these predictions and identify new mir. This strategy combined with comparative sequence analysis identified 129 sequences that corresponded to mature microRNAs (miR). A total of 107 sequences aligned to known human mir, and 100 of these matched expressed miR. The other seven sequences represented novel miR expressed from the complementary strand of previously characterized human mir. The 22 sequences without matches displayed characteristic mir secondary structures when folded in silico, and 10 of these retained sequence conservation with other vertebrate species. Expression analysis based on sequence identity counts revealed that some miR were preferentially expressed in certain tissues, while bta-miR-26a and bta-miR-103 were prevalent in all tissues examined. These results support the premise that species differences in regulation of gene expression by miR occur primarily at the level of expression and processing.