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Data de publicació electrònica: 09-04-2021 ; The area of the Spanish Pyrenees is particularly interesting for studying the demographic dynamics of European rural areas given its orography, the main traditional rural condition of its population and the reported higher patterns of consanguinity of the region. Previous genetic studies suggest a gradient of genetic continuity of the area in the West to East axis. However, it has been shown that micro-population substructure can be detected when considering high-quality NGS data and using spatial explicit methods. In this work, we have analyzed the genome of 30 individuals sequenced at 40× from five different valleys in the Spanish Eastern Pyrenees (SEP) separated by less than 140 km along a west to east axis. Using haplotype-based methods and spatial analyses, we have been able to detect micro-population substructure within SEP not seen in previous studies. Linkage disequilibrium and autozygosity analyses suggest that the SEP populations show diverse demographic histories. In agreement with these results, demographic modeling by means of ABC-DL identify heterogeneity in their effective population sizes despite of their close geographic proximity, and suggests that the population substructure within SEP could have appeared around 2500 years ago. Overall, these results suggest that each rural population of the Pyrenees could represent a unique entity. ; OL, IM, RT, JC, and SB acknowledge the support from Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, CERCA Program/Generalitat de Catalunya, Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, Generalitat de Catalunya through Departament de Salut and Departament d'Empresa i Coneixement, Co-financing with funds from the European Regional Development Fund by the Spanish Ministry of Science and Innovation corresponding to the Programa Operativo FEDER Plurirregional de España (POPE) 2014–2020 and by the Secretaria d'Universitats i Recerca, Departament d'Empresa i Coneixement of the Generalitat de Catalunya corresponding to the Programa Operatiu FEDER de Catalunya 2014–2020. OL gratefully acknowledges the financial support from Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness)—RYC-2013-14797, BFU2015-68759-P and PGC2018-098574-B-I00 and Generalitat de Catalunya (Government of Catalonia)—GRC 2017 SGR 937. IM gratefully acknowledges the financial support from the Government of Catalonia | Agència de Gestió d'Ajuts Universitaris i de Recerca (Agency for Management of University and Research Grants)—GRC 2014 SGR 615. MMA was supported by a FPU15/01251 from Ministerio de Ciencia, innovación y Universidades. PM and MMA acknowledge the financial support from Precipita-FECYT (FBG 309307 project). The samples analyzed in this study are part of a much larger sample whose collection was supported by the project CGL2011-27866 of the Ministerio de Ciencia y Tecnologia, the Fundació Moret i Marguí, and the invaluable collaboration of the Hospital San Bernabé (Berguedà), Fundació Sant Hospital (Alt Urgell), Hospital d'Olot i Comarcal (Garrotxa), Hospital de Campdevànol (Ripollès), and Hospital Comarcal (Pallars)