The objective was to study the persistence of drug resistance mutations detected earlier at virological failure during second or third line antiretroviral therapy. Therefore, in HIV-1 infected patients, with a virological treatment failure, genotypic resistance testing was carried out before change of therapy and at the next treatment failure. The majority of primary and secondary resistance mutations persisted in both the reverse transcriptase (RT) and the protease genes. After changing from zidovudine- to stavudine-containing regimens, the thymidine analogue mutations (especially M41L and T215Y/F) were found at new treatment failure in almost all patients. The M184V mutation disappeared in most (64%) non-3TC treated patients, although it persisted in a few didanosine- and abacavir-treated subjects. The primary protease inhibitor (PI) mutations reverted back to wild type in most patients who did not receive a new PI. In contrast, after changing from indinavir to saquinavir or nelfinavir, the M46I/L and/or V82A/F/ST disappeared in only 9 of 21 occasions at the new treatment failure. Most secondary mutations persisted with the exception of N88D. In patients with multiple treatment failures, most NRTI mutations thus persist frequently at new failures with modified treatment. A similar pattern is seen for protease inhibitors. The data suggest that clinical cross-resistance may develop via common pathways within all categories of drugs in heavily treated patients.