Gram-negative bacteria account for up to 35% of postoperative sternal wound infections (SWI) in patients undergoing cardiac surgery. Despite this, risk factors for Gram-negative SWI have not been investigated. The objective of this study was to define risk factors associated with Gram-negative SWI in patients undergoing cardiac surgery. 2590 patients undergoing cardiac surgery between 2002-2005 were prospectively monitored for development of SWI. Patient, operative, and post-operative risk factors were compared among patients that developed Gram-negative SWI and Gram-positive SWI to uninfected controls using univariate and multivariate analysis. A p < 0.05 was considered significant. Surgical site infections developed in 152 (5.9%) patients. Isolates were recovered from the sternum for 128 (5.0%) patients, from the leg donor site for 19 (0.73%) patients, and from the sternum and donor site for 5 (0.19%) patients. Gram-positive pathogens were isolated from 83 (3.3%) patients, Gram-negative pathogens from 42 (1.6%) patients, and mixed pathogens from 27 (1.0%) patients. Hospital admission greater than 48 hours before surgery (OR: 2.25; 95% CI: 1.11 - 4.58), ventilator-dependency preoperatively (OR: 5.32 95% CI: 2.22 - 12.75), and thoracentesis procedure postoperatively (OR: 3.71; 95% CI: 1.45 - 9.49) and diabetes (OR: 2.04; 95% CI: 1.17 - 3.55) were identified as significant risk factors for SWI due to Gram-negative bacteria using multivariate logistic regression. Diabetes, increased age, and peripheral vascular disease were identified as significant risk factors for SWI due to Gram-positive bacteria (p < 0.05, each). The risk factors associated with Gram-negative SWI differed significantly from those associated with Gram-positive SWI. Risk factors associated with Gram-negative SWI were identified. Unique interventions may be necessary to prevent Gram-negative SWI in cardiac surgery patients.