Carboxyhemoglobin levels in blood reflect endogenous carbon monoxide production and are often measured during routine blood gas analysis. Endogenous carbon monoxide production has been reported to be increased during sepsis, but carboxyhemoglobin levels have not been thoroughly evaluated as a biomarker of sepsis. We sought to determine whether carboxyhemoglobin levels were elevated during sepsis in a high risk population of premature neonates. We conducted a retrospective cohort study of 30 infants in two neonatal intensive care units using electronic medical and laboratory records. The majority of infants were extremely premature and extremely low birth weight, and 25 had at least one episode of sepsis. We collected all carboxyhemoglobin measurements during their in-patient stay and examined the relationship between carboxyhemoglobin and a variety of clinical and laboratory parameters, in addition to the presence or absence of sepsis, using linear mixed-effect models. We found that postnatal age had the most significant effect on carboxyhemoglobin levels, and other significant associations were identified with gestational age, hemoglobin concentration, oxyhemoglobin saturation, and blood pH. Accounting for these covariates, there was no significant relationship between the onset of sepsis and carboxyhemoglobin levels. Our results show that carboxyhemoglobin is unlikely to be a clinically useful biomarker of sepsis in premature infants, and raise a note of caution about factors which may confound the use of carbon monoxide as a clinical biomarker for other disease processes such as hemolysis.