Human immunodeficiency virus type 1 (HIV-1) transmitted drug resistance (TDR) is an important public health issue. In Brazil, low to intermediate resistance levels have been described. We assessed 225 HIV-1 infected, antiretroviral naïve individuals, from HIV Reference Centers at two major metropolitan areas of Sao Paulo (Sao Paulo and Campinas), the state that concentrates most of the Brazilian Aids cases. TDR was analyzed by Stanford Calibrated Population Resistance criteria (CPR), and mutations were observed in 17 individuals (7.6%, 95% CI: 4.5%–11.9%). Seventy-six percent of genomes (13/17) with TDR carried a nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutation, mostly K103N/S (9/13, 69%), potentially compromising the preferential first-line therapy suggested by the Brazilian HIV Treatment Guideline that recommends efavirenz-based combinations. Moreover, 6/17 (35%) had multiple mutations associated with resistance to one or more classes. HIV-1 B was the prevalent subtype (80%); other subtypes include HIV-1 F and C, mosaics BC, BF, and single cases of subtype A1 and CRF02_AG. The HIV Reference Center of Campinas presented more cases with TDR, with a significant association of TDR with clade B infection ( ).