Elevated levels of plasma mitochondrial DNA (mtDNA) have been reported in trauma patients and may contribute to the systemic immune response. We sought to determine the plasma levels of mtDNA in emergency department (ED) patients with and without sepsis and evaluate their association with severity of illness. This was a prospective observational study of patients presenting to one of three large, urban, tertiary care EDs. Patients were enrolled into one of three cohorts: (i) sepsis defined as suspected infection and two or more systemic inflammatory response criteria without hypotension, (ii) septic shock defined as sepsis plus hypotension despite an adequate fluid challenge, and (iii) control defined as noninfected ED patients without systemic inflammatory response/hypotension. Plasma levels of three mtDNAs were measured using real-time quantitative polymerase chain reaction. Levels of mtDNAs were compared among the three cohorts, and linear regression was used to assess the association between mtDNAs, interleukin 6, interleukin 10, and Sequential Organ Failure Assessment (SOFA) scores in patients with sepsis. We enrolled 93 patients: 24 control subjects, 29 patients with sepsis, and 40 patients with septic shock. As expected, comorbidities and SOFA score increased across categories. We found no difference in mtDNA levels between the three groups (P = 0.14-0.30). Among patients with sepsis, we found a small but significant negative association between mtDNA level and SOFA score, most clearly with cytochrome b (P = 0.03). We found no difference in mtDNA levels between control subjects and patients with sepsis. Mitochondrial DNA levels were negatively associated with organ dysfunction, suggesting that plasma mtDNA does not significantly contribute to the pathophysiology of sepsis.