HIV-1 molecular epidemiology studies carried out in Rio de Janeiro, Brazil have identified the prevalence of subtypes B, F1 and BF1 recombinants. A high percentage of HIV-1 subtype B isolates in Rio de Janeiro harbor the GWG motif at the V3 tip (B'' variant) instead of the canonical GPG motif (B variant). To trace the dynamics of the HIV-1 variants over time in different exposure categories in Rio de Janeiro, the HIV-1 proviral DNA from heterosexual men (HET) and men who have sex with men (MSM) from two distinct time periods (1990-1992 and 2008-2010) were extracted, and the env-gp120 region was amplified. Neighbor-joining phylogenetic analysis was performed to determine the viral subtype, and Bayesian analysis was used to trace the HIV-1 transmission networks. A predominance of subtype B was observed in both study periods, independent of the exposure risk category. An increase of non-B subtypes was observed in the HET group, but these subtypes were maintained among the MSM group. The distribution of HIV-1 subtype B signatures in the first and second periods studied were, respectively, HET (GPG) [44.8-51.5%], (GWG) [13.8-33.3%], and (GXG) [41.4-15.2%] and MSM (GPG) [34-50%], (GWG) [55.3-30.6%], and (GXG) [10.7-19.4%]. In the first period, an association between GWG and MSM was verified while a significant reduction of this association was observed during the second period. The phylogenetic tree and the BaTS program detected the clustering of isolates only according to the B signatures but not by exposure risk category. Our findings indicate a stable prevalence of HIV-1 subtypes B and F over time in Rio de Janeiro and further suggest that the B'' subclade of subtype B was possibly introduced into the MSM group in this area of Brazil.