An otolith shape and morphometric analysis was performed on European eel (Anguilla anguilla) subpopulations from five rivers and three coastal lagoons of Sardinia (central-western Mediterranean) to assess the role of different habitats on otolith development. Sagittal otolith shape was described by 11 harmonics from elliptic Fourier descriptors. Comparisons among the harmonics were run through canonical discriminant analyses (CDAs). The CDA reclassification rate (75.7%) demonstrated a spatial environmental discrimination among local eel subpopulations of Sardinia. The Euclidean distance values demonstrated a dissimilarity between the river and lagoon groups. The form factor and roundness shape indices were significantly higher in the river group than in the lagoon group. The distances of the first three rings to the otolith core revealed site-specific otolith development. Moreover, the annual otolith growth rate was faster in the lagoon group than in the river group. The differences among the studied sites in terms of sagittal otolith shape could relate to changes in different local stocks potentially related to environmental peculiarities. Establishing a direct correlation between otolith morphology and environmental factors is challenging, and further studies are needed to investigate the relationship between habitat type/environmental variation and growth/body characteristics of eels. Nevertheless, the achieved results suggest that this method can be considered to be a valuable tool for studying the ontogeny of the European eel.