2Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania 3Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan 4Muhimbili National Hospital, Dar es Salaam, Tanzania HIV human immunodeficiency virus type I (HIV-1) entry inhibitor potency is dependent on viral co-receptor tropisms and thereby tropism determination is clinically important. However, phenotypic tropisms of HIV-1 non-B subtypes have been poorly investigated and the genotypic prediction algorithms remain insufficiently validated. To clarify this issue, we recruited 52 treatment-naïve, HIV-1-infected patients in Tanzania, where multiple HIV-1 non-B subtypes co-circulate. Sequence analysis of 93 infectious envelope clones isolated from their plasma viral RNA revealed the co-circulation of subtypes A1, C, D, and inter-subtype recombinant forms (isRFs). Phenotypic tropism assays revealed that lentivirus reporters pseudotyped with 75 (80.6%) and 5 (5.4%) envelope clones could establish infection toward U87.CD4 cells expressing CCR5 (R5) and CXCR4 (X4), respectively; whereas the remaining 13 (14%) clones could infect both cells. Genotypic analyses by widely used algorithms including V3 net charge, Geno2pheno, WebPSSM, and PhenoSeq showed that almost all phenotypic X4-tropic clones and only 15 of 75 phenotypic R5-tropic clones were concordantly predicted. However, the remaining 60 phenotypic R5-tropic clones were discordantly predicted by at least one algorithm. In particular, 2 phenotypic R5-tropic clones were discordantly predicted by all algorithms tested. Taken together, the results demonstrate the limitation of currently available genotypic algorithms for predicting co-receptor inference among co-circulating multiple non-B subtypes and emerging isRFs. Also, the phenotypic tropism dataset presented here could be valuable for retraining of the widely used genotypic prediction algorithms to enhance their performance.