A digoxigenin (DIG)-based amplified fragment length polymorphism (AFLP) method was developed to distinguish three closely related citrus cultivars, ‘Kishu’, ‘Mukaku-Kishu’ and ‘Hira-Kishu’. In particular, ‘Mukaku-Kishu’ characterized by seedless fruit is very important for breeding purposes. Initial attempts at using the DIG-based RAPD procedure failed to detect ‘Mukaku-Kishu’-specific bands. However, the DIG-based AFLP method enabled to detect such bands, indicating that this method is more sensitive than the DIG-based RAPD procedure. The detailed protocol for the DIG-based AFLP methods is presented in this paper.