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Repeat blood cultures are frequently obtained in children with persistent fever and neutropenia (FN), but their clinical impact is uncertain.We identified children with persistent FN in the context of hematologic malignancy or hematopoietic stem cell transplantation from July 2006 to June 2012. For each episode, we reviewed blood cultures to determine the yield of true positive and false positive results. We then examined episode-level and culture-level predictors to determine factors associated with new bloodstream infections (BSI).Among 135 children who met inclusion criteria, there were 184 persistent FN episodes, during which 17 new BSI were diagnosed after the first 24 hr of fever (9.2%; 95% CI 5.4–15.3%). After the first 24 hr, the incidence of new BSI was 1.5% (95% CI 1.0–2.4%) per day and the incidence of blood culture contamination was 1.1% (95% CI 0.6–2.1%) per day. Of 17 new BSI identified, 14 (82%) required changes in therapy, while all 12 contaminant blood cultures were followed by additional antibiotic therapy. Increased odds of new BSI were associated with a history of BSI within 30 days of the episode (OR 5.18; 95% CI 1.29–20.8) and increasing time between recurrent fevers (OR 1.29; 95% CI 1.06–1.57).Repeat blood cultures have an important role in diagnosing new BSI and directing therapy in children with persistent FN. The current strategy could be improved by reducing the frequency of blood cultures after the first 24 hr, and targeting repeat cultures by risk. Pediatr Blood Cancer 2015;62:1421–1426. © 2015 Wiley Periodicals, Inc.