RDP3 is a new version of the RDP program for analyzing recombination events in DNA sequence alignments. It includes four new recombination analysis methods (3SEQ, VISRD, PHYLRO, and LDHAT), new tests for recombination hotspots, and various matrix methods for visualizing recombination patterns. It also allows for recombination-aware ancestral sequence reconstruction. RDP3 has a highly interactive graphical user interface that enables users to cross-check results with various phylogenetic tree construction and recombination signal visualization methods. It can analyze large datasets, including alignments up to 20 × 2 megabase sequences, within 48 hours on a desktop PC. RDP3 is available for free from its website and includes a detailed manual describing its methods and usage. RDP3 is a computer program for statistical identification and characterization of historical recombination events. It uses a range of non-parametric recombination detection methods to analyze aligned nucleotide sequences and provides detailed breakdowns of recombination breakpoints and the identities of recombinant and parental sequences. It enables users to save edited sequence alignments with recombinant sequences removed or split into their constituent parts. A key strength of RDP3 is its ability to detect recombination events in any part of a dataset, not just between reference strains or species. This makes it suitable for analyzing complex recombinants. However, the flexibility of RDP3 can make it difficult to assess the uncertainty of inferred recombination patterns. RDP3 complements probabilistic recombination analysis approaches with a wide range of cross-checking tools. RDP3 includes new features such as three new non-parametric recombination detection methods, a parametric recombination rate estimation method, two new tree construction methods, two recombination hotspot tests, a test for recombination-induced protein misfolding, and various matrix methods for visualizing recombination patterns. It also automatically scans alignments for recombination signals and infers the minimum number of recombination events needed to account for these signals. RDP3 uses heuristic methods to identify recombinant sequences and automatically checks for sequence misalignment, which can cause false positive recombination signals. RDP3 is highly optimized for speed and can analyze datasets with up to 40 million nucleotides within 48 hours on a standard 2 GHz processor with 2 GB of RAM. It is funded by various organizations, including the Wellcome Trust and the European Research Council. No conflicts of interest are declared.RDP3 is a new version of the RDP program for analyzing recombination events in DNA sequence alignments. It includes four new recombination analysis methods (3SEQ, VISRD, PHYLRO, and LDHAT), new tests for recombination hotspots, and various matrix methods for visualizing recombination patterns. It also allows for recombination-aware ancestral sequence reconstruction. RDP3 has a highly interactive graphical user interface that enables users to cross-check results with various phylogenetic tree construction and recombination signal visualization methods. It can analyze large datasets, including alignments up to 20 × 2 megabase sequences, within 48 hours on a desktop PC. RDP3 is available for free from its website and includes a detailed manual describing its methods and usage. RDP3 is a computer program for statistical identification and characterization of historical recombination events. It uses a range of non-parametric recombination detection methods to analyze aligned nucleotide sequences and provides detailed breakdowns of recombination breakpoints and the identities of recombinant and parental sequences. It enables users to save edited sequence alignments with recombinant sequences removed or split into their constituent parts. A key strength of RDP3 is its ability to detect recombination events in any part of a dataset, not just between reference strains or species. This makes it suitable for analyzing complex recombinants. However, the flexibility of RDP3 can make it difficult to assess the uncertainty of inferred recombination patterns. RDP3 complements probabilistic recombination analysis approaches with a wide range of cross-checking tools. RDP3 includes new features such as three new non-parametric recombination detection methods, a parametric recombination rate estimation method, two new tree construction methods, two recombination hotspot tests, a test for recombination-induced protein misfolding, and various matrix methods for visualizing recombination patterns. It also automatically scans alignments for recombination signals and infers the minimum number of recombination events needed to account for these signals. RDP3 uses heuristic methods to identify recombinant sequences and automatically checks for sequence misalignment, which can cause false positive recombination signals. RDP3 is highly optimized for speed and can analyze datasets with up to 40 million nucleotides within 48 hours on a standard 2 GHz processor with 2 GB of RAM. It is funded by various organizations, including the Wellcome Trust and the European Research Council. No conflicts of interest are declared.