In Hong Kong, the CCR5 antagonist has recently been introduced into salvage therapy for multiclass drug-resistant HIV-1-infected patients. Coreceptor usage must be determined prior to the usage of the CCR5 antagonist, which does not inhibit X4-tropic viruses. This study aimed to determine the tropism prevalence for HIV-1 subtypes B and CRF01_AE in Hong Kong. In addition, a modified promoter-PCR phenotypic assay was used to validate the genotypic tropism prediction on CRF01_AE. One hundred and five subtype B and 98 CRF01_AE antiretroviral-naive patients were recruited for this study. The viral env V3 region isolated from the patients was sequenced and analyzed by Geno2pheno (FPR=5.75% or 10%, Clonal or Clinical), position-specific scoring matrix (WebPSSM, x4r5 subtype B matrix), and the combination of 11/25 and net charge rules. Fifteen concordant and 22 discordant tropism genotyped CRF01_AE samples were further phenotyped by either enhanced sensitivity Trofile assay or an optimized promoter-PCR phenotypic assay. The prevalence of Dual/Mixed- or X4-tropic virus in antiretroviral-naive subtype CRF01_AE was 39.1%, which was significantly higher than subtype B (p<0.05), regardless of the choices of genotypic algorithms. Our phenotypic data proposed that a better genotypic tropism prediction for HIV-1 CRF01_AE would be using both Geno2pheno (FPR=10%, Clonal) and WebPSSM (x4r5 subtype B matrix) algorithms in combination. The sensitivity and specificity for this combination were 88.9% and 89.3%, respectively. The comparatively high prevalence of Dual/Mixed- or X4-tropic virus in CRF01_AE demonstrated the need for special attention to future treatment strategies.