Only a small portion of human immunodeficiency virus type 1 (HIV-1) particles entering the host cell results in productive infection, emphasizing the importance of identifying the functional virus population. Because integration of viral DNA (vDNA) is required for productive infection, efficient vDNA detection is crucial. Here, we use click chemistry to label viruses with integrase coupled to eGFP (HIVIN-eGFP) and visualize vDNA. Because click labeling with 5-ethynyl-2'-deoxyuridine is hampered by intense background staining of the host nucleus, we opted for developing HIV-1 reverse transcriptase (RT)-specific 2'-deoxynucleoside analogs that contain a clickable triple bond. We synthesized seven propargylated 2'-deoxynucleosides and tested them for lack of cytotoxicity and viral replication inhibition, RT-specific primer extension and incorporation kinetics in vitro, and the capacity to stain HIV-1 DNA. The triphosphate of analog A5 was specifically incorporated by HIV-1 RT, but no vDNA staining was detected during infection. Analog A3 was incorporated in vitro by HIV-1 RT and human DNA polymerase γ and did enable specific HIV-1 DNA labeling. Additionally, A3 supported mitochondria-specific DNA labeling, in line with the in vitro findings. After obtaining proof-of-principle of RT-specific DNA labeling reported here, further chemical refinement is necessary to develop even more efficient HIV-1 DNA labels without background staining of the nucleus or mitochondria.