To document the actual genetic diversity of HIV-1 strains in the Republic of Congo, 114 HIV-1 positives persons were sampled in 2003 and 2004 after their informed consent. They were attending the teaching hospital, the reference health center in Makelekele, Brazzaville and the regional hospital centers in Pointe-Noire, Gamboma and Ouesso. A total of 104 samples were genetically characterized by direct sequencing of the p24 gag region and 80 were also subtyped in the V3-V5 env region. The genetic subtype distribution of the Congolese strains showed the predominance of subtype A (36.5% and 32.5% in gag and env, respectively) and G (30.8% and 21.25%), whereas subtype D strains represented 12.5% and 15%. Subtypes C, F, H, J, K and the CRFs-01, -02, -05 -06, and also the recently characterized CRF18 were seen at lower rates. Finally, 4.8% (gag) and 6.25% (env) of the strains could not be classified. Moreover, a high intra-subtype diversity was observed in our study. Among 70 strains which have been characterized in the two genomic regions, 14 (20%) appeared to be unique recombinants. These data show a high genetic variability in the Republic of Congo, where all the subtypes have been documented together with certain subsubtypes and several CRFs.