We evaluated morphological differentiation among populations of threespine stickleback, Gasterosteus aculeatus, from the Limia and Mio basins (Galicia, NW Spain), the river Karup (Denmark) and Aiguamolls de l’Empord (Girona, northeast Spain), using multivariate analysis of morphometric and meristic characters. Multivariate discriminant analysis and hierarchical cluster analysis uncovered significant differences among the four main groups, between the two groups from Galicia and among populations within the Mio basin. The stickleback from Galicia differed from the Danish group in head and body armour characters. Between the Mio and Limia groups, we found differences in both head and body proportions as well as in number of gill rakers. Within the Mio basin populations varied with respect to head and body armour traits and the number of lower gill rakers. Clustering analysis divided these populations into two groups based on the latter trait: fish from the upper section of river Mio and tributaries (a lower number of lower gill rakers) and fish from the central and lower reaches of the river. Overall, morphometric traits were more useful than meristic ones; however, the two types of data provided congruent information about the morphological differentiation of stickleback populations.