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Introduction: In Brazil, the use of antiretrovirals is widespread: more than 260,000 individuals are currently undergoing treatment. We conducted a survey targeting antiretroviral‐naïve individuals who were initiating antiretroviral therapy (ART) according to local guidelines. This survey covered five Brazilian regions. Methods: The HIV Threshold Survey methodology (HIV‐THS) of the World Health Organization was utilized, and subjects were selected from seven highly populated cities representative of all Brazilian macro‐regions. Dried blood spots (DBS) were collected on SS903 collection cards and were transported by regular mail at room temperature to a single central laboratory for genotyping. Results: We analysed samples from 329 individuals initiating highly active antiretroviral therapy (HAART), 39 (11.8%) of whom were harbouring transmitted drug resistance (TDR). The mean CD4+ T cell count was 253 cells/µL, and the mean viral load was 142,044 copies/mL. The regional prevalence of resistance was 17.0% in the Northeast, 12.8% in the Southeast, 10.6% in the Central region, 8.5% in the North and 8.5% in the South. The inhibitor‐specific TDR prevalence was 6.9% for nucleoside reverse transcriptase inhibitors, 4.9% for non‐nucleoside reverse transcriptase inhibitors and 3.9% for protease inhibitors; 3.6% of individuals presented resistance to more than one class of inhibitors. Overall, there were trends towards higher prevalences of subtype C towards the South and subtype F towards the North. Of the DBS samples collected, 9.3% failed to provide reliable results. Discussion: We identified variable TDR prevalence, ranging from intermediate to high levels, among individuals in whom HIV disease progressed, thus implying that resistance testing before initiating ART could be effective in Brazil. Our results also indicate that the use of DBS might be especially valuable for providing access to testing in resource‐limited and remote settings., Introduction As of December 2012, more than 260,000 individuals were receiving antiretroviral treatment in Brazil, accounting for almost two‐thirds of the individuals living with HIV who are followed by the [...]