Mob proteins from distantly related eukaryotic species share very high sequence similarity and they are characteristic of a conserved Mob domain with around 180 amino-acid residues in length. However, the evolutionary relationship of mob family genes has not been extensively investigated. Through a phylogenetic approach, we have conducted a comprehensive evolutionary analysis of the mob gene family. Here we show that over 270 mob family members from protists to animals can be organized in four distinct groups. This classification is strongly supported by the analysis of mob exon-intron structures. Moreover, the conservation and divergence patterns of different groups of Mob proteins have been elucidated. Structural information and the identification of fixed amino acid substitutions provide evidence about the putative significance of specific residues in the structural integrity and/or molecular functions of Mob proteins. Thus, this study reveals the evolutionary history of mob gene family and provides a basis for functional studies of Mob proteins