Human populations relied on fish for food for thousands of years, as evidenced in numerous Palaeolithic archaeological sites across Europe. A better understanding of past aquatic ecosystems provides insights into fish ecology and evolution, the impact of past climate changes on intraspecific biodiversity, and ultimately, human reliance on fish resources in the past.In this study, we investigated past populations of brown trout (Salmo trutta L.) along the Bay of Biscay coast before, during, and after the Last Glacial Maximum (LGM), 33–9 kyr B.P. and compared their phenotypic diversity with modern populations. A total of 270 salmonid vertebrae from 11 archaeological sites in Spain and France were analysed. Species identification, combining ZooMS analyses with morphological traits and vertebral body size, confirmed 218 specimens as brown trout. A model for Salmonids species identification based on vertebral size is presented. Stable isotope analyses of carbon and nitrogen revealed a continuum of ecotypes. After testing multiple published regression models for body length estimation in brown trout, we developed a novel linear regression model alongside a protocol for age estimation. Overall, the analysis of body size, age and migratory ecotype demonstrated that the phenotypic diversity of brown trout populations during the Pleistocene-Holocene transition is comparable to that of modern populations, reinforcing the role of the Iberian glacial refugium in maintaining diversity during the LGM. The presence of large freshwater fish in human-occupied archaeological sites further suggests the exploitation of both riverine and marine fish resources.