Otolith shape analysis is commonly used in fisheries sciences to validate the relationship with fish length, to discriminate between stock units of fish, and to identify fish species from archeological data or stomach contents. However, all otolith studies have used two-dimensional (2D) images, a partial representation of the whole shape of the otolith. This study presents a comparative analysis of 2D and three-dimensional (3D) otolith shape data for many commercial species (flatfish: Lepidorhombus whiffiagonis, Scophthalmus maximus, Scophthalmus rhombus, Solea solea, Limanda limanda, Microstomus kitt, Platichthys flesus, Pleuronectes platessa and roundfish: Clupea harengus, Sardina pilchardus, Sprattus sprattus, Engraulis encrasicolus, Gadus morhua, Melanogrammus aeglefinus, Merlangius merlangus, Trisopterus luscus, Mullus surmuletus, Eutrigla gurnardus, Chelidonichthys cuculus, Chelidonichthys lucerna, Trachurus trachurus, Zeus faber) in the Eastern English Channel and the North Sea. 2D otolith images were made with an optical scanner, and 3D otolith images with an X-ray microtomograph. For each species, several specimens were selected from the complete size range. Otolith shape was assessed by analyzing several descriptors from 2D and 3D images from the otolith size parameters (Length, Width, Perimeter, Area and Volume). The relationship between fish length and otolith shape parameters are not the same within species and/or among species for 2D versus 3D data. Moreover, the relationship between 2D otolith size parameters and 3D otolith parameters are different among species. These results demonstrate the importance of 3D otolith shape analysis. In the future, the number of studies using otolith shape analysis in three dimensions must be increased strongly to validate previous studies on 2D images.