FastME 2.0 is an advanced and comprehensive program for distance-based phylogeny inference, improving upon the Neighbor Joining (NJ) method by incorporating Subtree Pruning and Regrafting (SPR) topological moves. The program offers several features, including: 1. **Distance Estimation**: Support for various models and options for estimating evolutionary distances from DNA and protein sequences. 2. **Bootstrapping**: Implementation of Felsenstein's bootstrap method to assess tree reliability. 3. **Parallel Computing**: Parallelization for efficient processing of large datasets. 4. **User Interfaces**: Command-line, PHYLIP-like, and a web server interface for user convenience. 5. **Algorithms**: Multiple tree-building algorithms to optimize balanced minimum evolution (BME) and Ordinary Least Square minimum evolution (OLSME). FastME 2.0 is designed to be fast and accurate, providing substantial improvements over NJ in terms of tree quality and computational efficiency. The program is available as open-source C code and can be integrated into phylogenomics pipelines.FastME 2.0 is an advanced and comprehensive program for distance-based phylogeny inference, improving upon the Neighbor Joining (NJ) method by incorporating Subtree Pruning and Regrafting (SPR) topological moves. The program offers several features, including: 1. **Distance Estimation**: Support for various models and options for estimating evolutionary distances from DNA and protein sequences. 2. **Bootstrapping**: Implementation of Felsenstein's bootstrap method to assess tree reliability. 3. **Parallel Computing**: Parallelization for efficient processing of large datasets. 4. **User Interfaces**: Command-line, PHYLIP-like, and a web server interface for user convenience. 5. **Algorithms**: Multiple tree-building algorithms to optimize balanced minimum evolution (BME) and Ordinary Least Square minimum evolution (OLSME). FastME 2.0 is designed to be fast and accurate, providing substantial improvements over NJ in terms of tree quality and computational efficiency. The program is available as open-source C code and can be integrated into phylogenomics pipelines.