HIV-1 reverse transcriptase (RT) inhibitors form the major backbone of antiretroviral therapy (ART) for HIV treatment and prevention programs1. To fast-track elimination, WHO recommends a massive scale up of ART. However, increasing disease burden, drug resistance, as well as long term ART toxicity and intolerance, threaten these efforts with an eventual exhaustion of drug options predicted2. Despite these challenges, there is little in the way of new drug classes in the discovery pipeline, apart from those within pre-existing classes that are susceptible to cross-resistance3. This gap warrants an intensified search for new and better drug classes3. To this end, we have initiated a fragment-based drug discovery (FBDD) program targeting HIV-1 RT to identify inhibitory small molecular weight (MW<250 Da) compounds (i.e. “fragments”). HIV-1 RT is an ideal target for FBDD since it’s a conformationally flexible enzyme that is critical for viral replication4 and a validated drug target5. Fragments can be strategically elaborated into larger, higher affinity inhibitors4, while simultaneously probing novel druggable pockets for drug design. Here we used a combination of biophysical (SPR, X-ray crystallography) and biochemical functional (RT activity inhibition) assays to screen and characterise compound libraries for novel RT inhibitory fragments and new druggable allosteric sites. Our screen identified three hits that inhibited the polymerase activity of both wild-type and non-nucleoside RT inhibitor (NNRTI)-resistant HIV-1 RT in the micromolar range, with two of these exhibiting distinctive mechanisms compared to clinically available anti-HIV-1 drugs6. To advance hit-lead optimisation based on these parental scaffolds, we have established a preliminary structural activity relationship (SAR) profile based on ~ 240 functional isomers spanning the three parental fragment scaffolds. We have identified several hits with ~50-200-fold greater potency relative to the parent compounds, where SAR and molecular modelling have guided on-going organic synthesis of high potency lead molecules using medicinal chemistry.