Live-attenuated HIV-1 vaccines are considered unsafe because faster-replicating pathogenic virus variants may evolve after vaccination. As an alternative vaccine approach, we have previously presented a doxycycline (dox)-dependent HIV-1 variant that was constructed by incorporating the tetracycline-inducible gene expression system (Tet-On system) into the viral genome. Replication of this HIV-rtTA virus is driven by the dox-inducible transcriptional activator rtTA, and can be switched on and off at will. A large scale evolution study was performed to test the genetic stability of this conditional-live vaccine candidate. In several long-term cultures, we selected for HIV-rtTA variants that no longer required dox for replication. These evolved variants acquired a typical amino acid substitution either at position 19 or 37 in the rtTA protein. Both mutations caused rtTA activity and viral replication in the absence of dox. We designed a novel rtTA variant with a higher genetic barrier towards these undesired evolutionary routes. The corresponding HIV-rtTA variant did not lose dox-control in long-term cultures, demonstrating its improved genetic stability.