Although HIV-1 clade B variants are predominant in Western Europe, non-B subtypes are rapidly spreading, mainly due to immigration from endemic regions. All newly diagnosed HIV-1-infected individuals at a HIV/AIDS clinic in Madrid from 2000 to 2007 were identified. Subtype assignment was based on phylogenetic analysis of pol sequences from plasma specimens collected at first visit. A total of 1,430 newly diagnosed HIV-1 individuals were identified: 902 Spaniards, 232 South Americans, and 162 Africans, among others. The proportion of South-Americans and Africans among diagnosed HIV-1 patients increased from 2000 to 2007 (from 17% to 22% and from 4% to 21%, respectively). Half of diagnosis of HIV-1 in 2007 was in foreigners whereas in previous years Spaniards were predominant. Non-B variants were found in 157 (24%) of the 649 subjects who could be subtyped: 11A, 6C, 2D, 1F2, 13G, 4H, 1J, 3CRF01_AE, 64CRF02_AG, 2CRF03_AB, 3CRF06_cpx, 3CRF10_CD, 7CRF11_cpx, 9CRF12_BF, 9CRF14_BG, 1CRF18_cpx, 1CRF19_cpx, 2CRF31_BC, 10 URF and 5 outgroups. They represented 93%, 14% and 4% of newly-diagnosed HIV-1 Africans, South-Americans and native Spaniards, respectively. Non-B subtypes increased from 9% in 2000 to 32% in 2007, specially among South-Americans (from 11% to 20%) and native Spaniards (from 4% to 10%). Most (75%) were recombinant viruses. The highest number and diversity of HIV-1 variants among natives was observed in 2007. HIV-1 non-B subtypes are increasingly present among newly diagnosed HIV-1 individuals in Madrid, representing a third of cases in 2007, whereas 10% of newly diagnosed HIV-1 native Spaniards had non-B viruses.