iTree is an open-source phylogenomic pipeline, which automates the execution of phylogenetic analyses under multithreaded and grid-computing environments, providing a scalable high-throughput platform for performing genome-wide evolutionary analyses. Furthermore, we describe the results of two applications of using iTree: (1) taxonomic assignment of 16S ribosomal RNA sequences from human oral metagenomic samples and (2) detection of horizontal gene transfer in microbial genomes.