Abstract The European anchovy (Engraulis encrasicolus) is one of the most important species in fisheries, representing the majority of landings worldwide. Identification of genetic stocks and assessment of divergence among them, is critical in order to implement management strategies. Population genetic structure in the Mediterranean basin is not clear and has not been extensively investigated with highly informative markers for population analyses. In this work, we aimed to identify a small SNP panel to be utilized for fine scale population genetic analysis within the Mediterranean basin. In order to do that, we used a set of 424 species-specific SNPs for assessing differentiation among E. encrasicolus populations within the Mediterranean and between Atlantic and Mediterranean Sea. Hence, we applied a Fst ranking method, for selecting a SNP sub-set from the large 424 SNPs panel and we compared the results obtained with the two sets of markers. Population assignment power and patterns of population differentiation were comparable. Analyses revealed a clear distinction between anchovies in the Atlantic and Mediterranean areas, and lower differentiation among Mediterranean populations. Our approach was successful in selecting a 96 SNP subset with high resolution and cost effectiveness to genotyping that can represent a useful tool for population genetic studies and stock management in this economically important species.