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Objectives The aim of the study was to determine the modifications of the mutational archive in proviral HIV-1 DNA occurring during 24 months of intermittent or continuous highly active antiretroviral therapy (HAART). Methods The study population included subjects enrolled in the Istituto Superiore di Sanità Pulsed Antiretroviral Therapy (ISS PART) clinical trial. All of these patients were on first-line HAART and had plasma HIV-1 RNA below 50 HIV-1 RNA copies/mL. A genotypic resistance test was performed on HIV-1 DNA extracted from peripheral blood mononuclear cells (PBMC) at baseline and after 24 months of follow-up. Resistance-associated mutations (RAMs) were defined according to the International AIDS Society (IAS) USA classification. Results Sixty-nine subjects were included in the study [36 enrolled in arm A of the ISS PART (continuous HAART) and 33 enrolled in arm B (intermittent HAART)]. No major modifications of the mutational archive were found in either group after 24 months of follow-up, in terms of both the proportion of subjects with mutations and the total number of mutations. Conclusions In this patient population, the mutational archive in HIV-1 DNA extracted from PBMC was stable for 24 months, irrespective of HAART modality, whether continuous or intermittent. [ABSTRACT FROM AUTHOR]