AbstractSummaryiTOL is a powerful and comprehensive phylogenetic tree visualization engine. However, adjusting to new templates can be time-consuming, especially when many templates are available. We developed an R package namely itol.toolkit to help users generate all 23 types of annotation files in iTOL. This R package also provides an all-in-one data structure to store data and themes, accelerating the step from metadata to annotation files of iTOL visualizations through automatic workflows.Availability and implementationThe manual and source code are available athttps://github.com/TongZhou2017/itol.toolkitTwo roads to add box plot annotation into a phylogenetic tree for iTOL.