Person who inject illicit substances have an important role in HIV-1 blood and sexual transmission and together with person who uses heavy non-injecting drugs may have less than optimal adherence to anti-retroviral treatment and eventually could transmit resistant HIV variants. Unfortunately, molecular biology data on such key population remain fragmentary in most low and middle-income countries. The aim of the present study was to assess HIV infection rates, evaluate HIV-1 genetic diversity, drug resistance, and to identify HIV transmission clusters in heavy drug users (DUs). For this purpose, DUs were recruited in the context of a Respondent-Driven Sampling (RDS) study in different Brazilian cities during 2009. Overall, 2,812 individuals were tested for HIV, and 168 (6%) of them were positive, of which 19 (11.3%) were classified as recent seroconverters, corresponding to an estimated incidence rate of 1.58%/year (95% CI 0.92–2.43%). Neighbor joining phylogenetic trees from env and pol regions and bootscan analyses were employed to subtype the virus from132 HIV-1-infected individuals. HIV-1 subtype B was prevalent in most of the cities under analysis, followed by BF recombinants (9%-35%). HIV-1 subtype C was the most prevalent in Curitiba (46%) and Itajaí (86%) and was also detected in Brasília (9%) and Campo Grande (20%). Pure HIV-1F infections were detected in Rio de Janeiro (9%), Recife (6%), Salvador (6%) and Brasília (9%). Clusters of HIV transmission were assessed by Maximum likelihood analyses and were cross-compared with the RDS network structure. Drug resistance mutations were verified in 12.2% of DUs. Our findings reinforce the importance of the permanent HIV-1 surveillance in distinct Brazilian cities due to viral resistance and increasing subtype heterogeneity all over Brazil, with relevant implications in terms of treatment monitoring, prophylaxis and vaccine development.