SUMMARY: High-throughput sequencing technologies have enabled cross-species comparative transcriptomic studies; however, there are numerous challenges for these studies due to biological and technical factors. We developed CoSIA (Cross-Species Investigation and Analysis), a Bioconductor R package and Shiny app that provides an alternative framework for cross-species transcriptomic comparison of non-diseased wild-type RNA sequencing gene expression data from Bgee across tissues and species (human, mouse, rat, zebrafish, fly, and nematode) through visualization of variability, diversity, and specificity metrics. AVAILABILITY AND IMPLEMENTATION: https://github.com/lasseignelab/CoSIA.