Abstract: Objective To construct NL4-3 env-EGFP/DsRed2 chimeric provirus and observe the fitness of HIV-1 CRF01-AE, CRF07/08-BC and B' subtype strains in vitro. Methods Gene env was amplified from 5 dried blood samples (DBS) which were infected by Thai-B', CRF07/08-BC, CRF01-AE subtype strains and 1 peripheral blood mononuclear cells (PBMC) sample which was infected by Zambia C subtype strain. Then 14 chimeric proviruses, NL4-3 env-EGFP/DsRed2 were constructed. Pair-wised competition assays were carried out to compare the fitness among all chimeric proviruses. Flow cytometry was used to identify the quantity of infected cells. Results Chimeric provirus from CRF01-AE subtype with relative fitness value (W) more than 1 showed greatest replicative fitness, followed by Thai-B, Zambia C, CRF07-BC/CRF08-BC (paired t-test, P0.05). Conclusion NL4-3 env-EGFP/DsRed2 can be used to compare the fitness of prevalent HIV strains in vitro by competition assay, and infer the impact of env gene on the HIV fitness.