Acute pharyngitis is a nonspecific symptom that can result from a number of viral or bacterial infections. For most etiologies, symptoms are self-limited and resolve without lasting effects; however, pharyngitis resulting from infection with Streptococcus pyogenes (a group A Streptococcus [GAS]) can be associated with serious sequelae, including acute rheumatic fever and acute glomerulonephritis. Rapid accurate detection of GAS in pharyngeal specimens from individuals suffering from pharyngitis aids in the management and selection of antibiotic therapy for these patients. A total of 796 pharyngeal swabs were collected at three separate clinical centers. Each specimen was analyzed using the illumigene group A strep DNA amplification assay (Meridian Bioscience Inc., Cincinnati, OH). To confirm GAS identification, the results were compared to those from direct and extracted culture methods using Gram staining and a GAS-specific latex agglutination test. Discrepant results were resolved using an alternative nucleic acid amplification test. The prevalence of culture-detected GAS in this study was 12.8% (102/796 specimens). The illumigene assay detected GAS in 74/74 direct culture-positive specimens (100% sensitivity) and 100/102 extracted culture-positive specimens (98.0% sensitivity). GAS was detected by the illumigene assay in an additional 42 specimens that were direct culture negative (94.2% specificity) and 16 specimens that were extracted culture negative (97.7% specificity). Discrepant analysis using an alternative molecular assay detected GAS nucleic acid in 13/16 (81.3%) false-positive specimens and 1/2 false-negative specimens, resulting in a final sensitivity of 99.0% and a specificity of 99.6% for the detection of GAS in pharyngeal swabs using the illumigene assay.