The critical role of the regulatory elements at the 5' end of the HIV-1 genome in controlling the life cycle of HIV-1 indicates that this region significantly influences virus fitness and its biological properties. In this study, we performed a detailed characterization of strain-specific variability of sequences from the U5 to upstream of the gag gene start codon of diverse HIV-1 strains by using next-generation sequencing (NGS) techniques. Overall, we found that this region of the HIV-1 genome displayed a low degree of intra-strain variability. On the other hand, inter-strain variability was found to be as high as that reported for gag and env genes (13-17%). We observed strain-specific single point and clustered mutations in the U5, PBS, and gag leader sequences (GLS), generating potential strain-specific transcription factor binding sites (TFBS). Using an infrared gel shift assay, we demonstrated the presence of potential TFBS such as E-box in CRF22_01A, and Stat 6 in subtypes A and G, as well as in their related CRFs. The strain-specific variation found in the sequence corresponding at the RNA level to functional domains of the 5' UTR, could also potentially impact the secondary/tertiary structural rearrangement of this region. Thus, the variability observed in this 5' end of the genomic region of divergent HIV-1 strains strongly suggests that functions of this region might be affected in a strain-specific manner. Our findings provide new insights into DNA-protein interactions that regulate HIV-1 replication and the influence of strain characterization on the biology of HIV-1 infection.