The remarkable viral diversity remains a big challenge to the development of HIV vaccines and optimal therapy worldwide. In the latest years, as a consequence of the large expansion of highly active antiretroviral therapy (HAART) availability worldwide, an increase in transmitted drug resistance mutations (TDRM) has been observed, varying according the region. This study assessed HIV-1 diversity and TDRM profile over time among newly HIV-1 diagnosed individuals from Rio de Janeiro, Brazil. Blood samples were collected from individuals seeking HIV diagnosis in four voluntary counseling and testing (VCTs) sites located in the Rio de Janeiro Metropolitan Area, in 2005-2007. Recent (RS) and long-term (LTS) HIV-1 seroconverters were distinguished using BED-CEIA. Pol viral sequences were obtained for 102 LTS identified in 2005 and 144 RS from 2005-2007. HIV-1 subtype and pol recombinant genomes were determined using Rega HIV-1 Subtyping Tool and by phylogenetic inferences and bootscanning analyses. Surveillance of HIV-1 TDRM to protease and reverse transcriptase inhibitors were performed according to the Calibrated Population Resistance (CPR) Tool 6.0. Overall, subtype B remains the most prevalent in Rio de Janeiro in both LTS and RS HIV-1 infected individuals. An increased proportion of recombinant samples was detected over time, especially in RS heterosexual men, due to the emergence of CRF02_AG and URF samples bearing a subtype K fragment. The prevalence of HIV-1 samples carrying TDRM was high and similar between LTS and RS (15.7% vs 14.6%) or age (<25yo 17.9% vs >25yo 16.6%) along the study period. The high resistance levels detected in both populations are of concern, especially considering the dynamics of HIV-1 diversity over time. Our results suggest that the incorporation of resistance testing prior to HAART initiation should be highly considered, as well as permanent surveillance, aiming to carefully monitoring HIV-1 diversity, with focus on CRF/URF emergence and putative transmission.