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Phenotypic differentiation among fish populations may be used for management of distinct stocks and helps in conserving biodiversity. We compared morphometric and meristic characters of the anchovy Anchoa januaria from shallow semi-closed bays between the south-eastern (Tropical, 23°S) and southern (Subtropical, 25°S) Brazilian coast. We hypothesized that differences between habitats and environmental conditions result in morphological divergence between conspecific populations. Fish size did not differ significantly between the two areas. Significant differences in the meristic and morphological characters were detected for individuals between the two areas, with specimens from the Subtropical region having significant larger head height, pectoral fin length and eye diameter compared with those from the Tropical region. Conversely, specimens from the Tropical region had significantly larger maxillary length, mouth length and body height than those from the Subtropical region. The number of rays for the dorsal and pectoral fins were higher for the specimens from the Subtropical region, whereas for the anal fin was higher for individuals from the Tropical region. Different morphological groups between the two regions were depicted by principal component analysis and discriminant function analysis, which suggest that morphological divergence is occurring. Local environmental influences and the lack of genetic interchange are likely to be the causes of such divergence. This is facilitated by the low tolerance of this species to marine waters that prevents connectivity between these stocks/populations.