OBJECTIVE Among 1222 antiretroviral-naive patients who received dolutegravir (DTG) as part of first-line therapy, none has developed resistance against this compound after 48-96 weeks of follow-up. Moreover, only four occurrences of virological failure with resistance mutations have been documented in previously drug-experienced patients who received DTG as a first time integrase inhibitor as a component of a second-line regimen. The R263K integrase resistance mutation was observed in two of these individuals who received suboptimal background regimens. We have previously selected mutations at position R263K, G118R, H51Y, and E138K as being associated with low-level resistance to DTG. Now, we sought to investigate the facility with which resistance on the part of R263K-containing viruses might develop. METHODS We tested the ability of DTG-resistant viruses containing either the R263K or G118R and/or H51Y mutations to develop further resistance against several reverse transcriptase inhibitors during in-vitro selection experiments. RESULTS Our results show that DTG-resistant viruses are impaired in their ability to acquire further resistance to each of nevirapine and lamivudine as a consequence of their relative inability to develop resistance mutations associated with these two compounds. CONCLUSIONS Our findings provide an explanation for the fact that no individual has yet progressed to virological failure with resistance mutations associated with dolutegravir in clinical trials in which patients received dolutegravir together with an optimized background regimen.