Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4(+) T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant virus’s RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.