Context .- Molecular diagnostics allow for rapid identification and detection of resistance markers of bloodstream infection, with a potential for accelerated antimicrobial optimization and improved patient outcomes. Although the impact of rapid diagnosis has been reported, studies in pediatric patients are scarce. Objective . -To determine the impact of a molecular blood-culture assay that identifies a broad-spectrum of pathogens and resistance markers in pediatric patients with gram-positive bloodstream infections. Design .- Data on the time to antimicrobial optimization, the length of hospitalization, and the hospital cost following implementation of a rapid assay were prospectively collected and compared with corresponding preimplementation data. Results . -There were 440 episodes from 383 patients included, 221 preimplementation episodes and 219 postimplementation episodes. Overall time to antimicrobial optimization was shortened by 12.5 hours (P = .006), 11.9 hours (P = .005) for bloodstream infections of Staphylococcus aureus specifically. Duration of antibiotics for those with probable blood-culture contamination with coagulase-negative staphylococci was reduced by 36.9 hours (P < .001). Median length of stay for patients admitted to general pediatric units was 1.5 days shorter (P = .04), and median hospital cost was $3757 (P = .03) less after implementation. For S aureus bloodstream infections, median length of stay and hospital cost were decreased by 5.6 days (P = .01) and $13 341 (P = .03), respectively. Conclusions .- Implementation of molecular assay for the detection of gram-positive pathogens and resistance markers significantly reduced time to identification and resistance detection, resulting in accelerated optimization of therapy, shorter length of stay, and decreased health care cost.