G-quadruplexes (GQs) play key regulatory roles within the human genome and have also been identified to play similar roles in other eukaryotes, bacteria, archaea, and viruses. Human immunodeficiency virus 1, the etiological agent of acquired immunodeficiency syndrome, can form two GQs in its long terminal repeat (LTR) promoter region, each of which act to regulate viral gene expression in opposing manners. The major LTR GQ, called LTR-III, is a distinct hybrid GQ containing a 12-nucleotide duplex loop attached to the quadruplex motif. The resulting quadruplex:duplex junction (QDJ) has been hypothesized to serve as a selective drug targeting site. To better understand the dynamics of this QDJ, we performed conventional and enhanced-sampling molecular dynamics simulations using the Drude-2017 force field. We observed unbiased and reversible formation of additional base pairs in the QDJ, between Ade4:Thy14 and Gua3:Thy14. Both base pairs were electrostatically favored, but geometric constraints within the junction may drive the formation of, and preference for, the Ade4:Thy14 base pair. Finally, we demonstrated that the base pairs are separated only by small energy barriers that may enable transitions between both base-paired states. Together, these simulations provide new insights into the dynamics, electrostatics, and thermodynamics of the LTR-III QDJ.