Patients with mitral valve prolapse (MVP) may reveal symptoms of autonomic dysfunction and heart rate variability (HRV). We sought to explore the autonomic nervous system in children with MVP. This cross-sectional study enrolled 60 children aged between 5 and 15 years with MVP and 60 age- and sex-matched healthy children as controls. Two cardiologists performed electrocardiography and standard echocardiography. HRV parameters were explored via 24-hour rhythm 3-channel Holter monitoring. The depolarization of ventricular and atrial parameters, comprising QT max and min, QTc intervals, QT dispersion, P maximum and minimum, and P-wave dispersion, was measured and compared. The mean age was 13.12±1.50 years in the MVP group (F/M: 34/26) and 13.20±1.81 years in the control group (F/M: 35/25). The maximum duration and P-wave dispersion in the MVP group were significantly different from the healthy children (P<0.001). The longest and shortest QT dispersion values and QTc values were significantly different between the 2 groups (P=0.004, P=0.043, P<0.001, and P<0.001, respectively). The HRV parameters were significantly different between the 2 groups, too. Decreased HRV and inhomogeneous depolarization showed that our children with MVP were prone to atrial and ventricular arrhythmias. Furthermore, P-wave dispersion and QTc could be used as prognostic markers of cardiac autonomic dysfunction before it is diagnosed by 24-hour Holter monitoring.