Background The main determinants of HIV-1 coreceptor usage are located in the V3-loop of gp120, although mutations in V2 and gp41 are also known. Incorporation of V2 is known to improve prediction algorithms; however, this has not been confirmed for gp41 mutations. Methods Samples with V3 and gp41 genotypes and Trofile assay (Monogram Biosciences, South San Francisco, CA, USA) results were taken from the HOMER cohort ( n=444) and from patients screened for the MOTIVATE studies ( n=1,916; 859 with maraviroc outcome data). Correlations of mutations with tropism were assessed using Fisher's exact test and prediction models trained using support vector machines. Models were validated by cross-validation, by testing models from one dataset on the other, and by analysing virological outcome. Results Several mutations within gp41 were highly significant for CXCR4 usage; most strikingly an insertion occurring in 7.7% of HOMER-R5 and 46.3% of HOM-ER-X4 samples (MOTIVATE 5.7% and 25.2%, respectively). Models trained on gp41 sequence alone achieved relatively high areas under the receiver- operating characteristic curve (AUCs; HOMER 0.713 and MOTIVATE 0.736) that were almost as good as V3 models (0.773 and 0.884, respectively). However, combining the two regions improved predictions only marginally (0.813 and 0.902, respectively). Similar results were found when models were trained on HOMER and validated on MOTIVATE or vice versa. The difference in median log viral load decrease at week 24 between patients with R5 and X4 virus was 1.65 (HOMER 2.45 and MOTIVATE 0.79) for V3 models, 1.59 for gp41-models (2.42 and 0.83, respectively) and 1.58 for the combined predictor (2.44 and 0.86, respectively). Conclusions Several mutations within gp41 showed strong correlation with tropism in two independent datasets. However, incorporating gp41 mutations into prediction models is not mandatory because they do not improve substantially on models trained on V3 sequences alone.