The aim of this study was to assess whether cardiac auscultation performed by pediatric trainees and neonatologists can reliably distinguish innocent from pathologic heart murmurs in asymptomatic neonates. From January 2008 to April 2009 the pediatric trainees of our institution were requested to refer all asymptomatic neonates with a murmur and classify the murmurs as "innocent," "pathologic," or "possibly pathologic." Prior to echocardiography, each neonate was evaluated by two experienced neonatologists. The echocardiographic studies of 169 neonates were analyzed. Abnormal cardiac anatomy was found in 55 neonates (32.6%). The overall ability of pediatric trainees in identifying congenital heart disease (CHD) was moderate [area under the curve (AUC) = 0.855] and significantly lower compared to neonatologists (AUC = 0.919, p = 0.007). However, at "lower" levels of clinical confidence (i.e., clinical diagnosis of possibly pathologic murmurs), pediatric trainees had good ability in excluding CHD (sensitivity 94.6%; negative likelihood ratio = 0.09). The ability of cardiac auscultation for diagnosing CHD in asymptomatic neonates is limited and dependent on the physician's experience and level of clinical confidence. Echocardiography should remain an option for all neonates with a possibly pathologic murmur.