Technical Abstract: Background: The southern cattle tick, Rhipicephalus (Boophilus) microplus, is an economically important parasite of cattle and can transmit several pathogenic microorganisms to its cattle host during the feeding process. Understanding the biology and genomics of R. microplus is critical to developing novel methods for controlling these ticks. Results: We present a global comparative genomic analysis of a gene index of R. microplus comprised of 13,643 unique transcripts derived from 42,512 expressed sequence tags (ESTs), representing a significant fraction of the complement of R. microplus genes. The source material for these ESTs consisted of polyA RNA from various tissues, lifestages, and strains of R. microplus, including larvae exposed to heat, cold, host odor, and acaricide. Functional annotation using RPSblast analysis identified conserved protein domains in the conceptually translated gene index and assigned GO terms to those database transcripts which had informative BlastX hits. Blast Score Ratio and SimiTri analysis compared the conceptual transcriptome of the R. microplus database to other eukaryotic proteomes and EST databases, including those from 3 ticks. The most abundant protein domains in BmiGI2 were also analyzed by SimiTri methodology. Conclusions: These results indicate that a large fraction of BmiGI entries have no homologs in other sequenced genomes. This highlights the important insights in tick biology which are likely to result from a tick genome sequencing project. Sets of genes were identified which had closer homologues to mammalian genes than to insect genes. Members of these groups would likely be poor choices as targets for development of novel tick control technology.