Phylogenomics, conventionally defined as the intersection of phylogenetics and genomics, has become a key instrument in a wide spectrum of biological studies, including resolution of complex evolutionary relationships, assignment of taxonomic affiliation, prediction of protein molecular functions, and tracing horizontal gene transfer event. Here, we introduce an open-source phylogenomic pipeline, iTree, 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.