Background: Sepsis represents a heterogeneous group of infectious etiologies. Early diagnosis and provision of appropriate antimicrobial therapy correlates with positive clinical outcomes. Current microbiological techniques are limited in diagnostic capacity and timeliness. Multiplex PCR has the potential to rapidly identify blood stream infections and fill this diagnostic gap. Methods: We identified patients from a large academic hospital emergency department with suspected sepsis. A Multiplex PCR that can detect 25 bacterial and fungal pathogens was compared to blood culture. Results were analyzed with respect to likelihood of infection, sepsis severity, site of infection, and the effect of prior antibiotic therapy. Results: We enrolled 306 subjects with suspected sepsis. Of these, 43 were later determined not to have infectious etiologies. Of the remaining 263 subjects, 70% had sepsis, 16% had severe sepsis and 14% had septic shock. The majority had definite (41.5%) or probable infection (30.7%). Blood culture and PCR performed similarly among patients with clinically defined infection (AUC 0.64 vs. 0.60, respectively). However, blood culture identified more cases of septicemia than did PCR among patients with an identified infectious etiology (66 vs. 46; p=0.0004). The two tests performed similarly when stratified by sepsis severity or infection site. Blood culture tended to detect infections more frequently among patients who received prior antibiotics (p=0.06). Conversely, PCR identified an additional 24 organisms that blood culture failed to detect. Conclusions: Real-time multiplex PCR has the potential to serve as an adjunct to conventional blood culture, adding diagnostic yield and shortening the time to pathogen identification.