As the emergence of highly resistant virus might compromise antiretroviral regimens in HIV-1 infected patients, a constant analysis of genotypic mutations should be performed to establish the magnitude of mutation prevalence and gauge their impact in patients treated extensively with combination therapy. The frequency of multiple dideoxynucleoside analogue resistance (MddNR) was evaluated in a group of Italian HIV-1 seropositive patients who failed to respond to therapy despite a long-lasting drug treatment. Results showed the presence of one or more mutations (A62V, V75I, F77L, F116Y and Q151M) able to confer resistance to all NRTIs in a relatively high percentage (7.9%) of patients enrolled in the study. Moreover, a significantly lower HIV-1 viral replication in patients with MddNR, suggested the importance of monitoring HIV-1 subjects not only by viral load, but also by drug resistance testing, so that a correct drug regimen may be chosen.