Gene transduction of hematopoietic progenitors capable of reconstituting both primary and secondary recipients is an important milestone in preclinical development of gene therapy. Myeloablation conditioning prior to infusion of transduced stem cells causes significant host morbidity. In contrast, drug-resistance gene transfer utilizes judicious in vivo selection of transduced stem cells over time, reaching only the level of transduction and expression required. The O⁶-benzylguanine (BG)-resistant mutant O⁶-methylguanine-DNA methyltransferase (MGMT) gene is a potent selection gene for transduced cells. Using two different mutant MGMTs, G156A and P140K, that vary in BG resistance by a factor of 1:20, we asked whether long-term repopulating and secondary mouse-repopulating cells could be transduced, transplanted, and selected for in the nonmyeloablated recipient and whether the mutant MGMT would continue to be expressed in secondary recipient repopulating cells. We found that under stringent drug-selection competition, cells expressing the more BG-resistant variant, P140K-MGMT, were enriched over G156A-MGMT-expressing progenitors. In addition, the MFG retroviral vector transmitted the mutant MGMT gene to long-term repopulating cells that, after selective enrichment in the nonmyeloablated primary recipient, repopulated secondary mice and continued to express the transgene. Thus, MFG mutant MGMT vectors transduce repopulating hematopoietic stem cells that may be used both for chemotherapeutic drug resistance and to enrich for second therapeutic genes.