European anchovy, Engraulis encrasicolus, is a small pelagic coastal marine fish largely spread from the North Sea to central Africa, including the entire Mediterranean and the Black and Azov Seas. The aim of the study is to understand the potential different population structure and their relationship between the Northeast Atlantic, Western, Eastern Mediterranean and the Black Sea anchovy population. The outlines of 2127 sagittal otoliths were collected from nineteen locations (English Channel, Atlantic, the Mediterranean and Black Sea), and added to existing information available from a previous study. Elliptical Fourier Analysis (EFA) was used to analyze the otolith shape variations among locations. Before examining the geographical differentiation by Linear discriminant (LDA) and Hierarchical Clustering Analysis, potentially confounding sources of variation (sex, fish length, otolith side and sampling year) were analyzed by partial RDA. Sex, sampling year and otolith side have no significant effect on the outline of otoliths. However, after taking into consideration the ontogenic effect, the geographical area has affects otolith shapes significantly. Three different groups of anchovies were identified: Atlantic-Southwestern Mediterranean, Northwestern-Eastern Mediterranean and Black Sea with a classification score of 80%. These results have implications for the stock management of anchovy populations from the North Sea to the Black Sea.