Background: Although the supine position remains the dominant position for external-beam partial breast irradiation (EB-PBI), the advantages of administering EB-PBI in the prone position have been recognized. The interobserver variability between target volumes delineated in the different positions for EB-PBI after breast-conserving surgery needs to be investigated. Methods: Twenty-seven patients suitable for EB-PBI were enrolled from July 2016 to April 2017. Supine and prone simulation CT images were sequentially acquired for all enrolled patients during free breathing. Five experienced radiotherapists delineated the target volumes for all patients on supine and prone simulation CT images. The selected parameters, including target volumes, the coefficient of variation (COV), the matching degree (MD), and so on, were calculated to analyze the interobserver variability. Results: Regardless of the patient position, the interobserver variability between tumor bed (TB) and clinical target volume (CTV) measurements in supine and prone positions were statistically significant (F = 31.34, 19.467; 44.000, 41.985; P = 0.000, 0.001; 0.000, 0.001). The interobserver variability of COV CTV was significantly greater in the supine position than in the prone position (T = 2.64, P = 0.014). Furthermore, the interobserver variabilities of MD TB and MD CTV were statistically lower in the supine position than in the prone position (Z = −3.460, −3.195, P = 0.000, 0.001). Conclusion: When delineating the target volume for EB-PBI, the interobserver variability in the prone position was lower than that in the supine position. Hence, the administration of EB-PBI in the prone position during free breathing is a reasonable option.