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Population structuring based on mitochondrial DNA variation along the rural landscape of the Trubia valley in North Iberia revealed significant association between road density and genetic distance between populations of two amphibian species, the midwife toad Alytes obstetricans and the palmate newt Lissotriton helveticus. Traffic calming (concentration of flows on minor rural roads at a few highways to decrease volumes and speeds) near urban settlements mitigates the population fragmentation of L. helveticus but not that of A. obstetricans, indicating that even small roads with low-intensity traffic act as barriers for the latter species. We suggest that the construction of passages for amphibians across rural roads would potentially mitigate population fragmentation of endangered species like the anuran A. obstetricans.