Abstract Background: Specific mutations in the reverse transcriptase (RT) gene of HIV-1 are associated with reduced activity of nucleoside inhibitors used in the antiretroviral treatment of infected patients. The appearance of these mutations may result in therapy failure. Therefore, HIV-1 genotyping is an important tool for monitoring antiretroviral therapy. At present different assay systems are used to obtain information about the changes in the viral genome. Objective: The aim of this study was to evaluate the LiPA HIV-1 RT assay version 1 for monitoring drug resistance mutations in comparison to full-length sequencing. Study design: Two hundred and forty-four samples were analyzed using the LiPA HIV-1 RT assay version 1 and were compared with full RT gene sequences obtained by in-house sequencing. Results: In 129/244 (52.9%) samples full concordance between both systems was found, in 86/244 (35.2%) samples at least one position was not detected by the LiPA assay, in 19/244 (7.8%) samples the results were contradictory, and in 10/244 (4.1%) contradictory as well as absent signals from the LiPA assay were found. Analyzing total codons, missing signals were observed at 137 codons, mainly found at positions 41 (40/137) and 215 (41/137). The 32 contradictions between LiPA and sequencing were equally distributed across all codons except for position 184 with only one case. The main reason for missing signals is the heterogeneity of the HIV genome, which could not be fully covered by the LiPA probes, e.g. unusual mutations or polymorphisms in the vicinity of the relevant positions. The same is the case for some contradictions, although most of them are not evident (19/32 positions). Conclusions: We analyzed a patient population with partly multiple therapy failures. The LiPA HIV-1 RT assay version 1 gives a high degree of samples with at least one missing signal (39.4%) in our cohort and this is not acceptable for a diagnostic tool. However, the LiPA assay might work better in untreated patients and could, therefore, still be used for screening.