FastTree 2 is an improved version of FastTree, a tool for inferring phylogenies from large alignments. It enhances accuracy without sacrificing scalability by incorporating minimum-evolution subtree-pruning-regrafting (SPRs) and maximum-likelihood nearest-neighbor interchanges (NNIs). FastTree 2 uses heuristics to restrict the search for better trees and estimates evolution rates using the "CAT" approximation. Despite being less accurate than methods using maximum-likelihood SPRs, FastTree 2 is significantly faster, completing the analysis of alignments with tens or hundreds of thousands of sequences in under a day on a desktop computer. For alignments with 500 sequences or more, FastTree 2 is at least 100 times faster than PhyML 3.0 and RAxML 7.2.1. FastTree 2 is suitable for bootstrapping and provides local support values based on the Shimodaira-Hasegawa test. It is freely available at http://www.microbesonline.org/fasttree.FastTree 2 is an improved version of FastTree, a tool for inferring phylogenies from large alignments. It enhances accuracy without sacrificing scalability by incorporating minimum-evolution subtree-pruning-regrafting (SPRs) and maximum-likelihood nearest-neighbor interchanges (NNIs). FastTree 2 uses heuristics to restrict the search for better trees and estimates evolution rates using the "CAT" approximation. Despite being less accurate than methods using maximum-likelihood SPRs, FastTree 2 is significantly faster, completing the analysis of alignments with tens or hundreds of thousands of sequences in under a day on a desktop computer. For alignments with 500 sequences or more, FastTree 2 is at least 100 times faster than PhyML 3.0 and RAxML 7.2.1. FastTree 2 is suitable for bootstrapping and provides local support values based on the Shimodaira-Hasegawa test. It is freely available at http://www.microbesonline.org/fasttree.