Background. The prevalence of drug resistance among persons with newly acquired human immunodeficiency virus (HIV) infection is well documented. However, it is unclear to what extent these mutations persist in chronically infected, treatment-naive patients. Methods. Prevalence of and factors associated with genotypic drug resistance were analyzed retrospectively in a subset of 491 chronically HIV-infected, antiretroviral-naive patients enrolled at 25 cities in the Terry Beirn Community Programs for Clinical Research on Acquired Immune Deficiency Syndrome (AIDS) Flexible Initial Retrovirus Suppressive Therapies trial during 1999-2001. Resistance was defined on the basis of the International AIDS Society 2003 definition, as well as the presence of additional mutations at codons 215 (C/D/E/S) and 69 (A/N/S) in the pol gene. Prevalence of mutations was estimated by use of techniques for stratified random samples. Logistic regression models were used to determine factors associated with resistance. Results. Among the 491 chronically HIV-infected patients (mean CD4 cell count, 269 cells/mm3; 31% of patients had a prior AIDS diagnosis), 57 (11.6%) had ≥1 resistance mutation, resulting in an estimated prevalence for the cohort of 10.8% (95% confidence interval [CI], 9.5%-12.1%). The prevalence was 8.8% if the 1181 mutation was excluded. By drug class, the estimated prevalence of mutations conferring resistance to nucleoside reverse-transcriptase inhibitors was 7.8%, and the prevalence was 3.0% for nonnucleoside reverse-transcriptase inhibitors and 0.7% for protease inhibitors. In a multiple logistic regression analysis, non-Hispanic white subjects were twice as likely than African American subjects to have resistance (odds ratio [OR], 2.1; 95% CI, 1.1-4.1; P = .03), and there was a 40% increase per year in prevalence of mutations by later year of enrollment (OR, 1.4; 95% CI, 1.0-2.1; P = .05). Conclusions. These results demonstrate the persistence of drug resistance mutations in chronically HIV-infected patients and an increasing prevalence of resistance over time, and they support genotyping of virus at baseline for chronically HIV-infected patients.