Recent development in high-throughput unbiased analytical tools (transcriptomics, proteomics and metabolomics) has opened the possibility to assess a multitude of molecular endpoints in organisms. In the present work, we used a combination of metabolomics tools, proton nuclear magnetic resonance spectroscopy (1H-NMR) and liquid chromatography coupled with mass spectrometry (LC–MS),) and multivariate statistical analysis to identify species-specific metabolome fingerprints and species-specific metabolites in the three Arctic copepods Calanus finmarchicus, C. glacialis and C. hyperboreus. Principal component analysis separated the three species with high specificity and sensitivity, and some species-specific metabolites were putatively annotated. These tools can be used for future studies within basal biology, systems biology, bioprospecting and ecotoxicology. As a supplementary analytical tool to genetic analyses, species-specific metabolites have a potential to be used to separate closely related Arctic Calanus species from net hauls.