The authors assessed HIV-1 variability in two distinct areas of Venezuela (the capital Caracas and Margarita Island) through the analysis of blood specimens and clinical and epidemiologic data of 72 persons. Proviral DNA was evaluated through heteroduplex mobility assay (HMA) based on the envelope region. Additionally, FOK I restriction enzyme digestion assay was performed in all subtype B ED31/33 amplified products to check the presence of the typical Brazilian subtype B GWGR variant. Sequencing and phylogenetic analysis for C2-V3 region of gp120 was performed in selected cases. The vast majority of samples were found to belong to subtype B, with a North American/European RFLP profile. An F subtype HIV-1 based on the region was identified for the first time in Venezuela. Genetic analyses of the protease and reverse transcriptase fragments of this sample depicted a recombinant B/F genetic profile. The discrimination capacity of HIV-1 subtypes using the primer set ED3/14-ED31/33 among the Caracas samples was found to be higher than for those from Margarita Island. The authors' results point to relevant differences between the samples of continental and Caribbean regions of Venezuela, requiring further evaluation of larger samples.