RDP3 is a new version of the RDP program for characterizing recombination events in DNA-sequence alignments. Among other novelties, this version includes four new recombination analysis methods (3SEQ, VISRD, PHYLRO and LDHAT), new tests for recombination hot-spots, a range of matrix methods for visualizing over-all patterns of recombination within datasets and recombination-aware ancestral sequence reconstruction. Complementary to a high degree of analysis flow automation, RDP3 also has a highly interactive and detailed graphical user interface that enables more focused hands-on cross-checking of results with a wide variety of newly implemented phylogenetic tree construction and matrix-based recombination signal visualization methods. The new RDP3 can accommodate large datasets and is capable of analyzing alignments ranging in size from 1000 × 10 kilobase sequences to 20 × 2 megabase sequences within 48 h on a desktop PC.