The Sine Oculis Homeobox (SIX) proteins play critical roles in organogenesis and are defined by the presence of two evolutionarily conserved functional motifs: a homeobox DNA binding domain and the SIX protein-protein interaction domain. Members of this transcription factor family can be divided into three subgroups: Six1/2, Six4/5, and Six3/6. This partitioning is based mainly on protein sequence similarity and genomic architecture, and not on specificities of DNA binding or binding partners. In fact, it is well demonstrated that members of the different subgroups can bind to and activate common transcriptional targets as well as form biochemical complexes with communal binding partners. Here we report that the C-terminal segment, which is not conserved across different SIX subfamilies, may serve to functionally distinguish individual SIX proteins. In particular, we have dissected the C-terminal region of Optix, the Drosophila ortholog of mammalian Six3/6, and identified three regions that distinguish Optix from Sine Oculis, the fly homolog of Six1/2. Two of these regions have been preserved in all Six3/6 family members while the third section is present only within Optix proteins in the Drosophilids. The activities of these regions are required, in unison, for Optix function. We suggest that biochemical/functional differences between members of large protein families as well as proteins encoded by duplicate genes can, in part, be attributed to the activities of nonconserved segments. Finally, we demonstrate that a subset of vertebrate SIX proteins has retained the ability to function during normal fly eye development but have lost the ability to induce the formation of ectopic eyes.