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The shape of sagitta otoliths was used to compare individuals of little tunny (Euthynnus alleteratus) harvested on board commercial fishing vessels from the coastal areas along the Eastern Atlantic, including the Mediterranean Sea. Fish sampling and selection was designed to cover possible seasonal changes and tuna size. The research encompassed both morphometric and shape analyses of left sagittal otoliths extracted of 504 fish specimens. Four shape indices (Circularity, Roundness, Rectangularity, and Form-Factor) were significantly different between two groups, showing a statistical differentiation between two clear spatial units. The degree of divergence was even more pronounced along the rostrum, postrostrum, and excisura of the generated otolith outlines between these two groups. One group corresponds to the samples from the coastal areas in the Northeast Temperate Atlantic and Mediterranean Sea (NETAM Area) and a second group from the coastal areas off the Eastern Tropical Atlantic coast of Africa (ETA Area). This study is the first to use otolith shape to differentiate tunas from separate spatial units. These results could be used to re-classify previously collected samples and to correct time series of data collected. [ABSTRACT FROM AUTHOR]