This work had the aim of comparing the method of hybridization with digoxigenin and the chemiluminescent detection system with the method of radioactive labeled. The cultivars used were susceptible Florunner and resistant COAN. The genomic DNA was isolated from young leaves and Southern blotting analysis was conducted using RFLP probe R2430E. The detection of the probe labeled with digoxigenin was as sensitive as labeled by the radioactivity, however with advantages above the method radioactive. Due to be a safe method and to present reduced costs, the demarcation method based on the digoxigenin and chemiluminescent detection is highly recommendable.