Human immunodeficiency virus (HIV)-1 reverse transcriptase (HIV-1 RT) is responsible for the transcription of viral RNA genomes into DNA genomes and has become an important target for the treatment of acquired immune deficiency syndrome (AIDS). This study used biophysical techniques to characterize the HIV-1 RT structure, monomer forms, and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) bound forms. Inactive p66_W401A and p51_W401A were selected as models to study the HIV-1 RT monomer structures. Nuclear magnetic resonance (NMR) spectroscopy revealed that the unliganded forms of p66_W401A protein and p51_W401A protein had similar conformation to each other in solution. The complexes of p66_W401A or p51_W401A with inhibitors showed similar conformations to p66 in the RT heterodimer bound to the NNRTIs. Furthermore, the results of paramagnetic relaxation enhancement (PRE)-assisted NMR revealed that the unliganded forms of the p66_W401A and p51_W401A conformations were different from the unliganded heterodimer, characterized by a greater distance between the fingers and thumb subdomains. Small-angle X-ray scattering (SAXS) experiments confirmed that p66_W401A and p51_W401A can bind with inhibitors, similar to the p66/p51 heterodimer. The findings of this study increase the structural knowledge base of HIV-1 RT monomers, which may be helpful in the future design of potent viral inhibitors.