We implemented the Representational Difference Analysis (RDA) screening method to identify genome variations between related bacteriophages without the need for complete genome sequencing. The strategy, optimized on phiKMV and LKD16 and further evaluated on the newly isolated phage LUZ19, is based on three successive rounds of reciprocal RDA, with an increasing driver/tester molar ratio from 100/1 to 750/1. Using three relevant restriction endonucleases, only 4 to 6 sequences per restriction enzyme are necessary to provide sufficient discriminatory information to reveal the major genome variations between phages.