The aim of this study was to evaluate the prevalence of genotypic resistance for each drug-class, and of single resistance-mutations in 1075 HIV-1 infected multi-treated patients undergoing their first genotypic resistance-test (GRT) after virological failure, over the years 1999-2003. First GRT was requested in 2003 for patients at earlier stages of failure, with less advanced disease, higher CD4-cell-count, lower HIV-RNA, and lower drug-experience with respect to 1999. Prevalence of resistance to all three drug-classes decreased from 33.3% in 1999 to 14.8% in 2003 (p < 0.001). Patients with protease-inhibitor (PI) resistant viruses decreased from 68.1% in 1999 to 34.1% in 2003 (p < 0.001); patients with nucleoside reverse transcriptase-inhibitor (NRTI) resistant viruses remained unchanged (85.4% in 1999; 86.4% in 2003); patients with non-NRTI (NNRTI) resistant viruses increased from 36.1% in 1999 to 52.3% in 2003 (p = 0.005) (corresponding to an increased NNRTI-use and decreased PI-use). From 1999 to 2003, resistance-mutations to drugs with high genetic-barrier significantly decreased (L90M/V82A/M46I/I54V/G73S/I84V/G48V for PIs; M41L/D67N/L210W/V1181 for NRTIs, p < 0.05), while mutations to drugs with low genetic-barrier increased (D30N in protease, M184V/K103N/V108I in reverse transcriptase, p < 0.05). Taken together, earlier recruitment to first GRT in patients with less severe disease, and with lower prevalence of drug-resistant viruses may further improve therapeutic strategies aimed at longer and greater control of HIV-related disease progression.