Coccidioides is a primary fungal pathogen of humans, causing life-threatening respiratory disease known as coccidioidomycosis (Valley fever) in immunocompromised individuals. Recently, Sharpton et al (2009) found that the deuterolysin (M35) family genes were significantly expanded in both the Coccidioides genus and in U. reesii, and that Coccidioides has acquired three more M35 family genes than U. reesii. In the present work, phylogenetic analyses based on a total of 28 M35 family genes using different alignments and tree-building methods consistently revealed five clades with high nodal supports. Interestingly, likelihood ratio tests suggested significant differences in selective pressure on the ancestral lineage of three additional duplicated M35 family genes from Coccidioides species compared to the other lineages in the phylogeny, which may be associated with novel functional adaptations of M35 family genes in the Coccidioides species, e.g., recent pathogenesis acquisition. Our study adds to the expanding view of M35 family gene evolution and functions as well as establishes a theoretical foundation for future experimental investigations.