The emergence of resistance-associated mutations to the antiretroviral agents and the genetic variability of HIV-1 impose challenges to therapeutic success. We report the results of genotype testing assays performed between 2002 and 2006 in 240 antiretroviral-experienced patients followed up in an HIV reference center in Brazil. Drug resistance mutations and viral subtypes were assessed through the algorithms from the Brazilian Genotyping Network (RENAGENO-Brazil) and from Stanford University. Mutation 184VI was the most prevalent (70%) and the thymidine analogue mutations that appeared most frequently were 215FY, 41L, 67N, and 210W, in this order. Among nonnucleoside reverse transcriptase inhibitor mutations, 103NS (32.5%) stood out. HIV subtype B was identified in 184 patients (76.7%). A significant increasing trend in the prevalence of non-B subtypes was observed during the study period (p=0.004). The main differences in prevalence of mutations among HIV-1 subtypes were related to viral protease, with 20MRI, 36I, and 89IMT more prevalent among non-B subtypes, and 84V, 10FR, 63P, 71LTV, and 77I more common in subtype B (p<0.05). Most mutations to etravirine had a prevalence lower than 10%, but at least one mutation to this drug was observed in 45% of the patients. In only 11 patients (4.6%) three mutations to etravirine were verified. Regional surveillance of the resistance profile and HIV-1 subtypes is crucial in the context of public health, to prevent the transmission of resistant strains and to guide the introduction of new drugs in a specific population.