Resistance to the first-generation non-nucleoside reverse transcriptase inhibitors nevirapine and efavirenz is characterized by rapid selection of viruses carrying one or several mutations in the reverse transcriptase gene, which immediately confer high-level resistance as well as cross-resistance to the two drugs. Such mutations have been detected close to the non-nucleoside reverse transcriptase inhibitor binding site and also in the connection domain of HIV reverse transcriptase. They lead to a loss of drug affinity without affecting viral fitness. As a single mutation is enough to confer high-level resistance, transmission of non-nucleoside reverse transcriptase inhibitor-resistant viruses (currently 2-7% of cases) is strongly associated with virologic failure of non-nucleoside reverse transcriptase inhibitor-based first-line regimens. The development of second-generation non-nucleoside reverse transcriptase inhibitors is a major challenge. The most promising compounds, etravirine and rilpivirine, are active on mutant viruses and possess a relatively high genetic barrier for resistance. Data on etravirine resistance in patients already exposed to first-generation non-nucleoside reverse transcriptase inhibitors show that, among 17 mutations in the reverse transcriptase gene, at least three must be present simultaneously in order to diminish etravirine activity. Recent studies of the prevalence of resistance in large databases of patients already exposed to nevirapine and efavirenz show that more than three-quarters of strains will still be sensitive to etravirine in both the southern and northern hemispheres. The first data on rilpivirine resistance are encouraging, but still too preliminary.