Human immunodeficiency virus type 1 (HIV-1) antiviral drug resistance is a major consequence of therapy failure and compromises future therapeutic options. Nelfinavir and lopinavir/ritonavir-based therapies have been widely used in the treatment of HIV-infected patients, in combination with reverse transcriptase inhibitors. The aim of this observational study was the identification and characterization of mutations or combinations of mutations associated with resistance to nelfinavir and lopinavir/ritonavir in treated patients. Nucleotide sequences of 1,515 subtype B HIV-1 isolates from 1,313 persons with different treatment histories (including naïve and treated patients) were collected in 31 Spanish hospitals over the years 2002-2005. Chi-square contingency tests were performed to detect mutations associated with failure to protease inhibitor-based therapies, and correlated mutations were identified using statistical methods. Virological failure to nelfinavir was associated with two different mutational pathways. D30N and N88D appeared mostly in patients without previous exposure to protease inhibitors, while K20T was identified as a secondary resistance mutation in those patients. On the other hand, L90M together with L10I, I54V, A71V, G73S, and V82A were selected in protease inhibitor-experienced patients. A series of correlated mutations including L10I, M46I, I54V, A71V, G73S, and L90M appeared as a common cluster of amino acid substitutions, associated with failure to lopinavir/ritonavir-based treatments. Despite the relatively high genetic barrier of some protease inhibitors, a relatively small cluster of mutations, previously selected under drug pressure, can seriously compromise the efficiency of nelfinavir- and lopinavir/ritonavir-based therapies.