Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, Ank^GAG1D4, showed a negative effect on the viral assembly of the HIV-1_NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of Ank^GAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-naïve HIV-1 infected individuals in northern Thailand during 2001⁻2012. SupT1, a stable T-cell line expressing Ank^GAG1D4 and ankyrin non-binding control (Ank^A32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/Ank^GAG1D4 demonstrated significantly lower levels of p24CA than SupT1/Ank^A32D3, which was found to correlate with the syncytia formation. This result suggests that Ank^GAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.