Purpose: Recent data indicate that neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C are valuable biomarkers for the differentiation and early diagnosis of acute kidney injury. We aimed to investigate that serum NGAL and cystatin C levels can be useful for differentiating between acute pyelonephritis (APN) and lower urinary tract infection (UTI) and predicting progress of renal scarring in pediatric UTI. Methods: This study was conducted at the Korea University Medical Center from November 2012 to July 2013. Eighty nine patients aged 1 month to 11 years admitted for a first UTI were enrolled. Serum NGAL and cystatin C levels were compared to clinical, laboratory, and radiologic findings retrospectively. Results: Mean serum NGAL concentration was significantly higher in patients with APN than in those with lower UTIs (APN vs. lower UTI, 473.97±74.45 ng/mL vs.126.45±27.23 ng/mL, p<0.001) while serum cystatin C level was not different between the two groups. Using a cutoff 233 ng/mL for serum NGAL for diagnosis of APN, sensitivity and specificity were 69.0% and 88.9%, respectively (area under the curve 0.856). The NGAL levels were strongly correlated with serum CRP concentrations (r=0.724, p<0.001), serum leukocyte counts (r=0.309, p<0.05), serum creatinine levels (r=0.412, p<0.001), fever duration (r=0.342, p<0.05), length of hospital stay (r=0.557, p<0.001), and the presence of initial renal cortical defect (r=0.605, p<0.001). Serum NGAL levels showed no correlation with the presence of veiscoureteral reflux and renal scarring, and serum cystatin C concentrations did not demonstrate any associations with these parameters. The mean NGAL levels in both APN and lower UTI groups were significantly higher before treatment than after treatment (p<0.05); however, mean cystatin C concentration in the lower UTI group was lower before treatment than after treatment (p<0.05). Conclusion: Serum NGAL can be a sensitive and specific biomarker to distinguish APN and lower UTI in pediatric UTI. However, serum cystatin C may be not useful for the prediction of APN in children with UTI.