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The diagnosis of pediatric septic arthritis (SA) can be challenging due to wide variability in the presentation of musculoskeletal infection. Synovial fluid Gram stain is routinely obtained and often used as an initial indicator of the presence or absence of pediatric SA. The purpose of this study was to examine the clinical utility of the Gram stain results from a joint aspiration in the diagnosis and management of pediatric SA.All patients with suspected SA who underwent arthrocentesis and subsequent surgical irrigation and debridement at an urban tertiary care children’s hospital between January 2007 and October 2016 were identified. Results of the synovial fluid Gram stain, as well as synovial cell count/differential and serum markers, were evaluated.A total of 302 patients that underwent incision and drainage for suspected SA were identified. In total, 102 patients (34%) had positive synovial fluid cultures and 47 patients (16%) had a microorganism detected on Gram stain. Gram stain sensitivity and specificity for the detection of SA were 0.40 and 0.97, respectively. This yielded a number needed to misdiagnose of 4.5 (ie, every fifth patient was misdiagnosed by Gram stain). For gram-negative organisms, the sensitivity dropped further to 0.13, with only 2/16 gram-negative organisms identified on Gram stain. Stepwise regression showed that age, serum white blood cell, and absolute neutrophil count were significant independent predictors for having a true positive Gram stain result. Elevated synovial white blood cell count was a significant predictor of having an accurate (culture matching the Gram stain) result.The Gram stain result is a poor screening tool for the detection of SA and is particularly ineffective for the detection of gram-negative organisms. The clinical relevance of the Gram stain and cost-effectiveness of this test performed on every joint aspiration sent for culture requires additional evaluation. Patients with gram-negative SA may be at high risk for inadequate coverage with empiric antibiotics due to poor detection of gram-negative organisms on initial Gram stain.Level III—case-control study.