RDP4 is the latest version of the Recombination Detection Program (RDP), a Windows-based software tool for detecting and visualizing recombination in virus genome sequences. It can analyze up to 2,500 sequences, each up to 10 Mb long, which is twice as many and three times longer than what older versions could handle. RDP4 is also applicable to bacterial full-genome datasets. It includes new features such as distinguishing recombination from genome segment reassortment, estimating recombination breakpoint confidence intervals, and various 'recombination aware' phylogenetic tree construction and comparison tools. It also provides new matrix-based visualization tools for examining individual recombination events and their overall phylogenetic impacts. Additionally, it includes new tests to detect the influence of gene arrangements, protein structure, nucleic acid secondary structure, nucleotide composition, and nucleotide diversity on recombination breakpoints. The key feature of RDP4 is its flexibility, allowing it to be run in fully automated mode or with a graphical user interface for detailed exploration of recombination events. RDP4 can detect recombination events without predefined non-recombinant reference sequences, making it more generally applicable. It uses various heuristic methods to identify recombination signals and then determines breakpoint positions using a hidden Markov model. It can then identify recombinant sequences using multiple methods. RDP4 can also infer the minimum number of recombination events needed to account for detected signals. It can be used to export sequence alignments with recombinant sequences removed or split into their constituent parts, which can then be used for more accurate evolutionary rate estimates or less error-prone positive selection inferences. RDP4 can also construct phylogenetic trees that account for detected recombination events and perform ancestral sequence inferences using various methods. RDP4 provides tools for analyzing overall recombination patterns, including tests for recombination hot and cold spots, tests of purifying selection against recombination-induced misfolding, tests of association between recombination breakpoints and genome features, and phylogenetic incompatibility visualizations. It can analyze datasets with up to 200 million nucleotides in 72 hours on a standard 2GHz processor with 2 GB of RAM. RDP4 is available for free download and includes a manual with detailed descriptions of its methods and usage instructions. It is also compatible with Mac and Linux computers.RDP4 is the latest version of the Recombination Detection Program (RDP), a Windows-based software tool for detecting and visualizing recombination in virus genome sequences. It can analyze up to 2,500 sequences, each up to 10 Mb long, which is twice as many and three times longer than what older versions could handle. RDP4 is also applicable to bacterial full-genome datasets. It includes new features such as distinguishing recombination from genome segment reassortment, estimating recombination breakpoint confidence intervals, and various 'recombination aware' phylogenetic tree construction and comparison tools. It also provides new matrix-based visualization tools for examining individual recombination events and their overall phylogenetic impacts. Additionally, it includes new tests to detect the influence of gene arrangements, protein structure, nucleic acid secondary structure, nucleotide composition, and nucleotide diversity on recombination breakpoints. The key feature of RDP4 is its flexibility, allowing it to be run in fully automated mode or with a graphical user interface for detailed exploration of recombination events. RDP4 can detect recombination events without predefined non-recombinant reference sequences, making it more generally applicable. It uses various heuristic methods to identify recombination signals and then determines breakpoint positions using a hidden Markov model. It can then identify recombinant sequences using multiple methods. RDP4 can also infer the minimum number of recombination events needed to account for detected signals. It can be used to export sequence alignments with recombinant sequences removed or split into their constituent parts, which can then be used for more accurate evolutionary rate estimates or less error-prone positive selection inferences. RDP4 can also construct phylogenetic trees that account for detected recombination events and perform ancestral sequence inferences using various methods. RDP4 provides tools for analyzing overall recombination patterns, including tests for recombination hot and cold spots, tests of purifying selection against recombination-induced misfolding, tests of association between recombination breakpoints and genome features, and phylogenetic incompatibility visualizations. It can analyze datasets with up to 200 million nucleotides in 72 hours on a standard 2GHz processor with 2 GB of RAM. RDP4 is available for free download and includes a manual with detailed descriptions of its methods and usage instructions. It is also compatible with Mac and Linux computers.