Abstract: The majority of mucosal HIV-1 infection is initially established by a few HIV-1 viral variants, followed by the development of overt systemic infection, and these viral variants are known as transmitted/founder viruses (T/F viruses). Investigation of the sensitivity of T/F virus to different anti-HIV-1 drugs will provide the best strategies of pre-exposure prophylaxis (PrEP) for high-risk groups of HIV-infected patients. Herein we constructed for the first time, a luciferase reporter system for HIV-1 T/F viruses, and then compared the drug sensitivity between T/F viruses and chronic infection virus. The result showed that the 50% inhibitory concentration (IC₅₀) of nucleoside reverse transcriptase inhibitors (NRTIs), integrase inhibitors (INIs) and protease inhibitors (PIs) were not significantly different between the T/F viruses and chronic infection viruses of the same subtype (P>0.05), while non-nucleoside reverse transcriptase inhibitors (NNRTIs) showed a moderate resistance to T/F viruses, with a significant increase in IC₅₀ (P<0.05). The conclusion suggests that when patients are in high-risk or in the acute infection of HIV-1, NNRTIs should be avoided in the first-line antiretroviral therapy regimens.