The objective of our study was to evaluate the performance of renal contrast-enhanced ultrasound (CEUS) against the 99m-labeled dimercaptosuccinic acid (DMSA) scan and computed tomography (CT) in children for the diagnosis of acute pyelonephritis. We included children who underwent both renal CEUS and the DMSA scan or CT. A total of 33 children (21 males and 12 females, mean age 26 ± 36 months) were included. Using the DMSA scan as the reference standard, the sensitivity, specificity, positive predictive value, and negative predictive value of CEUS was 86.8%, 71.4%, 80.5%, and 80.0%, respectively. When CT was used as the reference standard, the sensitivity, specificity, positive predictive value, and negative predictive value of CEUS was 87.5%, 80.0%, 87.5%, and 80.0%, respectively. The diagnostic accuracy of CEUS for the diagnosis of acute pyelonephritis was 80.3% and 84.6% compared to the DMSA scan and CT, respectively. Inter-observer (kappa = 0.54) and intra-observer agreement (kappa = 0.59) for renal CEUS was moderate. In conclusion, CEUS had good diagnostic accuracy for diagnosing acute pyelonephritis with moderate inter- and intra-observer agreement. As CEUS does not require radiation or sedation, it could play an important role in the future when diagnosing acute pyelonephritis in children.