Understanding connectivity between groups of a species is fundamental to effective management and conservation practices, yet is poorly understood for tropical estuarine fishes. Here, age-related trends in otolith elemental chemistry were examined to assess the degree of connectivity of Polydactylus macrochir, a large, non-diadromous, tropical estuarine teleost, across the species’ Australian distribution. Elemental signatures (7Li, 43Ca, 55Mn, 88Sr and 138Ba) of transverse sections of otoliths of 3+ yr fish from the 2005 year class collected from 17 locations were sampled using laser ablation inductively coupled plasma mass spectrometry, providing elemental profiles from the otolith core through the first 3 yr of a fish’s life. Univariate and cluster analyses revealed differences in elemental signatures of the otolith core among most locations, although similarities were evident among locations in the Gulf of Carpentaria and among 2 neighbouring locations in both Western Australia and the east coast of Queensland. Fewer differences were observed for post-settlement life history stages, although some differences were observed among neighbouring locations separated by as little as 50 km. SIMPROF analyses revealed that 138Ba generally provided the greatest discrimination among locations. Positive correlations were observed between otolith 138Ba concentration of individual fish and river flow indices for material laid down in all age groups, suggesting differences in flow was a significant driver of the observed patterns in 138Ba. Examined in conjunction with complementary studies into the connectivity of the study species, the spatial structuring suggests that P. macrochir populations are susceptible to localised depletion, with limited opportunity for replenishment from neighbouring populations. The results highlight the importance of using multiple, complementary methods for assessing connectivity of aquatic organisms.