Abstract Background Fostemsavir is a prodrug of a first-in-class HIV-1 attachment inhibitor, temsavir, that binds to gp120 and blocks attachment to the host-cell CD4 receptor, preventing entry and infection of the target cell. Previous studies using a limited number of clinical isolates showed that there was intrinsic variability in their susceptibility to temsavir. Objectives Here, an analysis was performed using all clinical isolates analysed in the Monogram Biosciences PhenoSense® Entry assay as part of the development programme. Methods In total, 1337 individual envelopes encompassing 20 different HIV-1 subtypes were examined for their susceptibility to temsavir. However, only seven subtypes (B, C, F1, A, [B, F1], BF and A1) were present more than five times, with subtype B (881 isolates) and subtype C (156 isolates) having the largest numbers. Results As expected, variability in susceptibility was observed within all subtypes. However, for the great majority of these viruses, temsavir was highly potent, with most viruses exhibiting IC50s <10 nM. One exception was CRF01_AE viruses, where all five isolates exhibited IC50s >100 nM. For the 607 isolates where tropism data were available, geometric mean temsavir IC50 values were remarkably similar for CCR5-, CXCR4- and dual mixed-tropic envelopes from infected individuals. Conclusions These data show that HIV-1 viruses from most subtypes are highly susceptible to temsavir and that temsavir susceptibility is independent of tropism.