Objectives The aim of the study was to gain more insight into the relationship between transmitted singletons found at HIV diagnosis by population sequencing and the possible presence of clinically relevant viral minorities containing additional resistance mutations. Methods We studied the viral quasispecies and therapy response in 10 individuals with transmitted single nucleoside reverse transcriptase inhibitor (NRTI)-related resistance mutations as detected by population sequencing. Results Ultra-deep pyrosequencing did not reveal additional drug-resistance mutations in nine of 10 patients. In these nine patients, no breakthrough with resistant viruses was observed despite the use of low genetic nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens in the majority of patients. Conclusions These data suggest that viral minority variants containing additional resistance mutations may be rare in patients with transmitted NRTI singletons in the Netherlands. Larger studies are required to confirm these findings and to determine the therapeutic consequences.