Previous studies have reported that patients with mitral valve prolapse (MVP) may display autonomic dysfunction. Measurement of heart rate variability (HRV) and P-wave dispersion (PWD) may provide insights into the functional state of the autonomic nervous system. Heart rate variability (HRV) has been used as a noninvasive marker of autonomic activity. However, to the authors’ knowledge, PWD has not been studied in the context of MVP. This study aimed to examine HRV and PWD in patients with MVP and to determine whether differences exist between symptomatic and asymptomatic patients. The study population consisted of 54 healthy children (17 boys and 37 girls) ages 6–18 years and 76 patients with MVP (20 boys and 56 girls) ages 6–18 years. The duration and dispersion of the P-wave were measured by surface 12-lead electrocardiograms (ECGs). Heart rate variability was quantified using both time-domain and frequency-domain analyses of Holter ECGs. The minimum duration of the P-wave was significantly lower in the MVP patients (42.4 ± 10.0 ms) than in the control subjects (54.4 ± 12.8 ms) (p < 0.01), and the PWD was significantly increased in the MVP group (42.7 ± 10.8 ms) compared with the control subjects (31.8 ± 10.9 ms) (p < 0.01). However, no significant differences were found between the symptomatic and asymptomatic patients. In addition, the HRV parameters were not statistically different between the two groups. In conclusion, although HRV parameters were not significantly different between the MVP and control groups, the findings show that PWD was increased for the children with MVP. However, no relationship could be established between PWD and clinical symptoms.