Primary HIV drug resistance, characterized by mutant virus strains in untreated HIV-infected persons, is of significant epidemiological significance. Primary resistance is associated with reduced efficacy of antiretroviral therapy (ART). We determined the prevalence of primary resistance in Nordrhein-Westfalen, Germany. Genotypic resistance testing was performed in a prospective multicenter study in chronically infected previously untreated HIV-positive patients before administration of first-line ART. Mutations were classified according to the International AIDS Society USA guidelines and the geno2pheno interpretation tool. Between January 2001 and December 2005, resistance testing was performed in 831 patients. 77.4% were males, the mean age was 39 years (SD: 10.5). The mean duration of diagnosis of HIV infection was 1.6 years (SD: 3.4). 32.4% of patients were at CDC stage C, mean CD4 cell count was 236 /microl (SD: 205), and mean viral load was 206,855 copies/ml (SD: 450,610). In total, resistance-associated mutations were detected in 75 patients (9.0%; 95%CI, 7.1-11.0). After inclusion of mutations E44D and V118I, resistance was identified in 99 patients (11.9%; 95%CI, 9.7-14.1). 5.4% had mutations indicating nucleoside reverse transcriptase inhibitor (NRTI) resistance (95%CI, 3.9-7.0), 3.0% had non-NRTI resistance (95%CI, 1.8-4.2), and 2.4% had protease inhibitor resistance (95%CI, 1.4-3.4), respectively. Two-class resistance was detected in 0.8% (95%CI, 0.2-1.5), three-class resistance in 0.5% (95%CI, 0.01-1.0). Mutations indicating revertant variants of resistant strains were found in 3.9% (95%CI, 2.5-5.2). Considering the variables age, gender, time since diagnosis, CDC stage, CD4 cell count, viral load, HIV subtype, ethnic origin, and HIV transmission group, no significant risk factor for the presence of primary resistance was demonstrated in univariate and mutlivariate analyses. The prevalence of primary resistant virus strains was about 10% in chronically infected ART-naive HIV-patients in the largest federal state of Germany. The majority of these patients had NRTI-associated resistance. No risk factor for the presence of primary drug resistance was identified. Because of the high prevalence and the possible impact on efficacy of drug treatment, routine genotypic resistance testing should be performed in untreated HIV-positive patients before administration of first-line ART.