While it is known that selection for specific HIV-1 drug resistance-associated mutations (DRM) occurs following ART failure, the patterns of resistance mutations, reduced susceptibility (RS), and replicative capacity (RC) that appear as the number of major NRTI mutations increases have been less well-studied. These changes were examined as a function of the number of major NRTI mutations using patient-derived HIV samples submitted for resistance testing between 2003-2005 (n = 35,222) that were grouped by number of NRTI-DRMs present. In the absence of NRTI-DRMs, few (3.4%) samples had RS to one or more NRTI, 33.6% to one or more NNRTI, and 12.6% to one or more PI. With one NRTI-DRM, 94% had RS to one or more NRTI, 50% to one or more NNRTI, and 33% to one or more PI. Increases in NRTI-DRMs were accompanied by increased prevalence of NNRTI and PI DRMs and RS. With one NRTI-DRM, the mean number of NRTIs with RS was 1.7, while when five NRTI-DRMs were present, RS to > or =5 NRTIs was observed. PI-DRM and RS increased at a slower rate than NNRTI-DRM and RS. RC declined from a mean of 97.8% for samples without NRTI-DRMs to 68.9% with one NRTI-DRM, possibly due to reduced fitness conferred by K65R or M184I/V, to an RC of 43.9% for samples with seven to eight NRTI-DRMs. The relatively high percent of samples with NNRTI-DRM but without NRTI-DRMs may result from selection following virologic failure, and/or transmission of virus uniquely resistant to NNRTI.