The retrotransposons that maintain telomeres in Drosophila melanogaster have unique features that are shared across all Drosophila species but are not found in other retrotransposons. Comparative analysis of these features provides insight into their importance for telomere maintenance in Drosophila. Gag proteins encoded by HeT-A(mel) and TART(mel) are efficiently and cooperatively targeted to telomeres in interphase nuclei, a behavior that may facilitate telomere-specific transposition. Drosophila virilis, separated from D. melanogaster by 60 MY, has telomeres maintained by HeT-A(vir) and TART(vir). The Gag proteins from HeT-A(mel) and HeT-A(vir) have only 16% amino acid identity, yet several of their functional features are conserved. Using transient transfection of cultured cells from both species, we show that the telomere association of HeT-A(vir) Gag is indistinguishable from that of HeT-A(mel) Gag. Deletion derivatives show that organization of localization signals within the two proteins is strikingly similar. Gag proteins of TART(mel) and TART(vir) are only 13% identical. In contrast to HeT-A, surprisingly, TART(vir) Gag does not localize to the nucleus, although TART(vir) is a major component of D. virilis telomeres, and localization signals in the protein have much the same organization as in TART(mel) Gag. Thus, the mechanism of telomere targeting of TART(vir) differs, at least in a minor way, from that of TART(mel). Our findings suggest that, despite dramatic rates of protein evolution, protein and cellular determinants that correctly localize these Gag proteins have been conserved throughout the 60 MY separating these species.