Background: Interest in co-receptor tropism (CRT) assays is growing with the availability of CCR5 antagonists for treatment of HIV-1 infection. The Trofile phenotypic assay is being used extensively to support the pre-clinical and clinical development of CRT inhibitors. The performance of genotype assays in heavily treatment-experienced patients - the likely target population for CCR5 antagonists - is unknown. Previous studies have highlighted technical difficulties in obtaining high quality envelope sequence and limitations of interpretation systems. Methods: Samples from triple-class experienced patients were studied (N ~600). CRT (R5, X4, or dual/mixed) was determined by the Trofile assay. Population V3 loop sequences were determined using conventional techniques. CRT was predicted (R5 only or X4 using) according to published algorithms: modified 11/25 rule, position-specific scoring matrix, decision trees, or a support-vector machine model, trained on publicly available data. Results: Based on Trofile results approximately half of the samples were defined as CXCR4-using; 25% had 6 or more mixtures in V3 (including variants with and without insertions or deletions) and were not interpretable. Another 25% had no mixtures in V3 at the protein level, and 50% had 1 to 5 mixtures in V3. Of the interpretable V3 sequences, overall concordance with phenotype ranged from 74 to 79%, and was characterized by high X4 specificity (90-100%) but low sensitivity (18-38%). Conclusions: In treatment-experienced patients, who are likely candidates for treatment with CCR5 antagonists, envelope sequencing is hampered by diversity in variable loop length amongst subpopulations of viruses in approximately one quarter of patients. When a V3 sequence can be unambiguously determined, state of the art interpretation algorithms significantly under-report the presence of X4-using virus.