The genus Wolbachia encompasses intracellular bacteria found in arthropods and in filarial nematodes. In arthropods, Wolbachia is primarily a reproductive parasite and shows relatively frequent horizontal transfer between host species, while in nematodes it appears to be a mutualist and is strictly vertically transmitted. We can expect that different selective pressures are acting on their genomes. Here we present an analysis of three Wolbachia genes, wsp, ftsZ and dnaA. In wsp of arthropod Wolbachia, an excess of non-synonymous substitutions was observed, providing evidence for positive selection. In nematode Wolbachia, no evidence for positive selection was found. Pressure for amino acid variation in wsp of arthropod Wolbachia could derive either from an arms race with the host or from the occurrence of more frequent hosts shifts due to horizontal transmission. In nematode Wolbachia, the lack of positively selected sites could result from the absence of an arms race, or from the homogeneity of the biochemical environment they exist in (ensured by strict vertical transmission). In ftsZ minor differences in substitution patterns were observed between arthropod and nematode Wolbachia, only in the 3'-portion of the gene. dnaA showed comparable patterns of variation in both lineages, with evidence for strong conservation.