We investigated HIV-1 reverse transcriptase (RT) polymorphisms of plasma isolates from 98 HIV-1-infected study subjects with >2 years of antiretroviral therapy who were failing their current protease inhibitor (PI)-containing regimen. In 1 patient, we detected a virus with a heavily mutated beta3-beta4 connecting loop of the HIV-1 RT fingers subdomain, consisting of a single aspartate codon insertion between positions 69 and 70 and five additional variations: 64N, K65, K66, 67G, 68Y, T69, Ins D, 70R, W71, R72, K73, 74I. Mutants with the recently described 2-aa insertions between codons 68 and 70 of RT were detected in another 3 patients. Among the four isolates with the 1- or 2-aa insertions, the novel genotype was the most refractory to therapy and displayed the highest level of phenotypic resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Follow-up samples demonstrated that the novel mutant represents a stable genetic rearrangement and that the amino acid insertions can coexist with nonnucleoside analogue reverse transcriptase inhibitors (NNRTI) mutations resulting in phenotypic resistance to both NRTIs and NNRTIs. An increasing number of HIV-1 isolates containing various insertions in the beta3-beta4 hairpin of the HIV-1 RT fingers subdomain appear to emerge after prolonged therapy with different NRTIs, and these polymorphisms can confer multiple drug resistance against NRTIs.