In this study, otolith morphology and otolith contour from big-scale sand smelt, Atherina boyeri were analyzed in Lake Eğirdir and Lake İznik (natural lakes), and Hirfanlı Dam Lake (artificial lake). The otolith contour and sixteen otolith morphological characters such as width, depth, shape, sulcus acusticus shape, ostium, cauda, rostrum size-shape, and thickness, antirostrum size-shape, and thickness, anterior and posterior regions, mesial and lateral surfaces were successfully investigated for three total length groups of A. boyeri inhabiting the lakes. Intraspecific variation in rostrum shape, rostrum size, rostrum thickness, antirostrum, anterior region, mesial and lateral surfaces were observed for the three total length groups of three A. boyeri populations. In the three different lakes, saccular otolith surface morphology of A. boyeri was detected such as a flattened surface for Lake Eğirdir, rough surface for Lake İznik, and relatively smooth surface for Hirfanlı Dam Lake using the Scanning Electron Microscope. The otolith contours showed noticeable differences in the three total length groups of A. boyeri from the different lakes. Intraspecific variability of the otolith characters and morphology consisting of the otolith surface morphology and otolith contour were presented and compared for the three total length groups of the A. boyeri from the different lakes.