The MEGA6 software, version 6.0, introduces new features for molecular evolutionary analysis, including the ability to infer timetrees using the RelTime method. This method estimates divergence times for all branching points in a phylogeny without requiring knowledge of lineage rate variation or clock calibrations. MEGA6 also includes a Timetree Wizard, a graphical user interface that simplifies the process of specifying phylogenies, sequence data sets, and calibration constraints. The software now supports both GUI and command-line versions, and is freely downloadable from www.megasoftware.net. MEGA6 enhances memory management, allowing it to handle larger sequence datasets. It also provides confidence intervals for divergence times, which are essential for assessing uncertainty in time estimates and testing biological hypotheses. The software uses a combination of variance contributions from lineage rate variation and estimated variance of relative node times to generate these confidence intervals. The RelTime method is efficient in terms of both performance and memory usage. For example, MEGA6 required only 43 minutes and 1 GB of memory to process a nucleotide alignment of 765 sequences and 2,000 bp. The software also allows users to convert relative times to absolute times using calibration points, and provides options for specifying calibration constraints. In addition to the new timetree system, MEGA6 includes enhancements such as the subtree-pruning-and-regrafting (SPR) and tree-bisection-and-regrafting (TBR) algorithms for searching optimal trees under maximum likelihood and maximum parsimony criteria. The software has also been upgraded to support 64-bit systems, allowing it to use up to 4 GB of memory. A usage analytics system has been implemented to assess the most commonly used options and analyses, helping to prioritize future developments. MEGA6 is supported by research grants from the National Institutes of Health and the Japan Society for the Promotion of Science. The software is widely used for comparative analyses of DNA and protein sequences to infer molecular evolutionary patterns of genes, genomes, and species over time.The MEGA6 software, version 6.0, introduces new features for molecular evolutionary analysis, including the ability to infer timetrees using the RelTime method. This method estimates divergence times for all branching points in a phylogeny without requiring knowledge of lineage rate variation or clock calibrations. MEGA6 also includes a Timetree Wizard, a graphical user interface that simplifies the process of specifying phylogenies, sequence data sets, and calibration constraints. The software now supports both GUI and command-line versions, and is freely downloadable from www.megasoftware.net. MEGA6 enhances memory management, allowing it to handle larger sequence datasets. It also provides confidence intervals for divergence times, which are essential for assessing uncertainty in time estimates and testing biological hypotheses. The software uses a combination of variance contributions from lineage rate variation and estimated variance of relative node times to generate these confidence intervals. The RelTime method is efficient in terms of both performance and memory usage. For example, MEGA6 required only 43 minutes and 1 GB of memory to process a nucleotide alignment of 765 sequences and 2,000 bp. The software also allows users to convert relative times to absolute times using calibration points, and provides options for specifying calibration constraints. In addition to the new timetree system, MEGA6 includes enhancements such as the subtree-pruning-and-regrafting (SPR) and tree-bisection-and-regrafting (TBR) algorithms for searching optimal trees under maximum likelihood and maximum parsimony criteria. The software has also been upgraded to support 64-bit systems, allowing it to use up to 4 GB of memory. A usage analytics system has been implemented to assess the most commonly used options and analyses, helping to prioritize future developments. MEGA6 is supported by research grants from the National Institutes of Health and the Japan Society for the Promotion of Science. The software is widely used for comparative analyses of DNA and protein sequences to infer molecular evolutionary patterns of genes, genomes, and species over time.