Codon usage bias is an effective measure of the differences among organisms at a genomic level. These genomic differences also reflect some differences in the organisms’ lifestyles and physiology. Here we demonstrate that prokaryotic obligate intracellular parasites and symbionts have a codon usage pattern that differs significantly from that of exclusively free-living prokaryotes. This result is valuable in that it suggests that the habitat of an organism may directly influence that organism’s use of synonymous codons, which in turn demonstrates evidence of an evolutionary mechanism that operates at a finer molecular level than that of amino acids and proteins.