The protease gene of human immunodeficiency virus type 1 (HIV-1) clinical isolates found in 15 immigrants (most of African origin) living in Spain was examined. Phylogenetic analyses were performed, taking as reference a panel of 26 HIV protease gene sequences deposited with GenBank. All specimens belonged to four distinct HIV-1 non-B subtypes: C (three cases), F (one), G (nine), and H (two). Five patients harboring subtype G strains were further classified within the IbNg recombinant clade. A high degree of genetic polymorphism at the protease gene was seen in all subtypes. Moreover, changes at positions associated with drug resistance were seen in subtype G viruses carried by patients who had not been exposed to protease inhibitors. Plasma viremia was lower than expected for some samples, according to the clinical features and the CD4+ cell count, suggesting that viral load titers were underestimated by all three commercially available techniques. This work represents the first genetic characterization of subtypes C, F, G, and H in Spain.