In this study, 27 HIV-1-positive patients on long-term highly active antiretroviral therapy (HAART) in the Czech Republic were followed for a period of up to 7 years. Variability of the HIV-1 protease (PR) sequence common in the Czech Republic was observed. Under the pressure of inhibitors of protease (PRIs) and reverse transcriptase (RTIs) mutations in PR were detected. Development of resistance to PRIs was followed by a decrease in CD4 count and increase in viral load. The dynamics of viral load closely corresponded to the accumulation of specific primary mutations in PR and RT. Out of 27 patients 18 developed resistance to PRIs and the prolonged therapy led to the accumulation of a higher number of amino acid changes associated with the resistance and, consequently, cross-resistance to several PRIs was observed. These multi-resistant variants of HIV-1 with mutations in PR could not be inhibited sufficiently with PRIs that are currently available in clinical practice. Efficient yet temporary suppression of viral replication was achieved by a lopinavir (LPV) treatment.