DSA after HTx may have adverse effects on patient survival. The aim of this study was to assess risk factors for the development of DSA after pediatric HTx. All HTx recipients at our center with serial monitoring of DSA were identified. Cox proportional hazards model was used to estimate donor and recipient characteristics associated with the development of DSA. De novo DSA were detected in 40 (33%) of 121 HTx recipients. Characteristics associated with de novo DSA included older age, African American race, prior operations, prior ECMO, PRA > 10%, longer bypass time, mechanical support at transplant, and donor death from GSW. In a multivariable model, mechanical support (HR 3.23, 95% CI [1.02, 8.87]), African American race (HR 3.36, 95% CI [1.68, 7.32]), and donor death from GSW (HR 4.76, 95% CI [1.62, 14.01]) were significantly associated with DSA. Multiple factors appear to play a role in the development of DSA, knowledge of which may guide the frequency of post-transplant monitoring. DSA develop more frequently in those with prior sensitizing events, suggesting the possibility that these exposures predispose the immune system to respond to donor antigens, even in the presence of a negative cross-match.