Otoliths are used in taxonomy and ichthyology as they can provide a wide range of information about specimens. They are an essential tool to monitor the most sensitive species for a sustainable exploitation level. Despite the increasing use of sagittae in research, their inter- and intra-specific variability and eco-functionality are still poorly explored. This paper aims to investigate the inter- and intra-specific variability of Mugilidae sagittae using morphological and morphometrical analysis, as well as scanning electron microscopy and shape analysis. The sagittae of 74 specimens belonging to three different Mugilidae species, collected from a coastal lagoon, were analyzed to give an accurate description of their morphology, morphometry, shape and crystalline habits. The results highlighted the intra- and inter-specific variability of sagittae, showing morphometrical differences among species and slight differences between left and right sagittae in C. labrosus individuals. Moreover, SEM images showed a peculiar crystal organization, with several different crystal habits and polymorphs. This study provides an accurate description of sagittae in the studied species, deepening the knowledge on inter- and intra-specific variations and crystal habits and providing data which will be useful for future studies on otoliths. With this data, it will be possible to improve conservation and exploitation sustainability in sensitive habitats.