Salvage therapy with efavirenz is often ineffective in patients having failed nevirapine treatment, even when mutations associated with efavirenz resistance are not detected by standard population-based genotyping. The presence of minority viral populations expressing efavirenz cross-resistance could explain these observations, and such populations were sought in plasma from patients failing nevirapine for whom genotyping revealed the presence of the Y181C mutation (usually associated with limited efavirenz cross-resistance) but not the K103N mutation (which produces high-level efavirenz resistance). Viral populations expressing K103N (>1% total virus) were detected by sequence-selective polymerase chain reaction in 4 of 16 patients failing nevirapine, although, in retrospect, the mutation was not perceptible in the original genotype in only 2 cases. Both patients with detectable K103N mutations who received efavirenz failed treatment, and virus expressing K103N emerged. Four of 5 patients without detectable K103N mutations also failed efavirenz, associated with the emergence of nonnucleoside reverse transcriptase mutations that included K103N in 2 cases. The emergence of a minority viral population expressing K103N was identified in 1 patient from a separate study group subsequent to discontinuing treatment with nevirapine. These findings support the idea that minority viral populations with distinct resistance genotypes, although undetectable by standard genotyping, can contribute to the failure of salvage regimens.