The structure of medusae populations is poorly known. Natural geochemical signatures based on elemental composition of calcified structures are a common tool for investigating population structure or connectivity in marine systems. Chironex fleckeri (Cubozoa) medusae have a hard calcified structure, the statolith. Laser ablation–inductively coupled plasma mass spectrometry was used to determine the elemental composition of statoliths at varying spatial scales. We investigated medusae population structure using both univariate (element:Ca ratios) and multivariate (multi-element:Ca signature) analyses. Significant differences in some elemental ratios were found among regions (separated by hundreds of kilometres) and among many sites (separated by kilometres) within regions. Canonical discriminant analyses of multi-element:Ca signatures successfully distinguished between both regions and sites within regions with correct classifications of 100% of samples to some locations. Statolith microchemistry can help discriminate populations of jellyfish, but a multiseason comparison demonstrated the need to calibrate spatial differences by season. Our evidence and recent ecological data suggest that populations of C. fleckeri medusae are highly localised at spatial scales of kilometres; potential causal factors are discussed.