Objective: To estimate HIV incidence and characterize subtype and resistance profile among recently diagnosed individuals from different at-risk populations from Argentina in the context of a Global Fund Project (Sub-project N°112).Methods: From October 2006 to September 2008, HIV prevalence surveys were conducted among men who have sex with men (MSM), sex workers (SWs), drug users (DUs) and pregnant women (PW). Volunteers older than 18 years old without previous HIV positive diagnosis were interviewed and tested for HIV. HIV positive samples were analyzed by the Standarized Testing Algorithm for Recent HIV Seroconversion to estimate incidence. By partial RT-PCR and sequencing of HIV pol gene antiretroviral resistance mutations were identified and HIV subtype was determined.Results: A total of 12,200 volunteers were incorporated to the cross-sectional HIV prevalence surveys. HIV prevalence was 10.4% (161/1549), 4.8% (24/473), 2.9% (7/241), 22.9% (52/227), 1.9% (21/1112) and 0.12% (10/8570) for MSM, Male DUs, Female DUs, Male SWs, Female SWs and PW, respectively. HIV incidence estimates were 6.36, 2.06, 1.39 and 0.05 per 100 person-years for MSM, DUs, SWs and PW, respectively. In SWs, a higher HIV incidence was found in men as compared with women (6.08 vs. 0.62 per 100 person-years). Phylogenetic analysis showed that 122 (61.6%) samples were subtype B, 74 (37.4%) were inter-subtype BF recombinants and 2 (1.0%) were subtype C. In all groups, men had higher prevalence of subtype B. HIV primary resistance mutations were found in 11.7% (23/198) without significant differences between groups. An association between B subtype and higher frequency of resistance mutations was found.Discussion: The high HIV prevalence, incidence and primary resistance mutations founds in this study call for an imperative need to have the Argentinean epidemic very well documented in order to continue and reinforce prevention strategies in the population under study.