Extracellular chemical cues constitute much of the language of life among marine organisms from microbes to mammals. These chemical cues shape foraging and feeding strategies, symbiotic interactions, selection of mates and habitats, and the transfer of energy and nutrients within and among ecosystems. Changes in the chemical pool flux within these ecosystems serve as invisible signals of overall ecosystem health and, conversely, disruption to this finely tuned equilibrium. In coral reef systems, the full scope and magnitude of the chemicals involved in maintaining reef equilibria are largely unknown. In particular, processes involving small, polar molecules, which form the majority components of labile dissolved organic carbon (DOC), are often poorly captured using traditional techniques and therefore remain unconstrained. In this study, we employed a recently developed chemical derivatization method and mass spectrometry-based targeted exometabolomics to capture and quantify polar dissolved phase metabolites on five coral reefs in the U.S. Virgin Islands. We detected and quantified 45 polar exometabolites, and further demonstrate their variability across the investigated geographic landscape and contextualize these findings in terms of geographic and benthic cover differences. By comparing our results to previously published coral reef exometabolomes that did not employ chemical derivatization, we show the novel quantification of 23 metabolites which were previously undetected in the coral reef exometabolome, including compounds involved in central carbon metabolism (e.g.,glutamate) and novel metabolites such as homoserine betaine. We highlight the immense potential of chemical derivatization-based exometabolomics for quantifying labile chemical cues on coral reefs and measuring molecular level responses to environmental stressors. Overall, improving our understanding of the composition and dynamics of reef exometabolites is vital for effective ecosystem monitoring and management strategies.SynopsisMinimal research exists on the polar exometabolites on coral reefs, an essential component of reef health and the majority component of labile DOC. This study reports 23 newly quantified polar metabolites on coral reefs and highlights significant differences in the exometabolome composition across geographically distinct reefs and bays.