The fragmentation of a population may have important consequences for population genetic diversity and structure due to the effects of genetic drift and reduced gene flow. We studied the genetic consequences of the fragmentation of the Spanish imperial eagle (Aquila adalberti) population into small patches through a temporal analysis. Thirty-four museum individuals representing the population predating the fragmentation were analysed for a 345-bp segment of the mitochondrial control region and a set of 10 nuclear microsatellite loci. Data from a previous study on the current population (N = 79) were re-analysed for this subset of 10 microsatellite markers and results compared to those obtained from the historical sample. Three shared mitochondrial haplotypes were found in both populations, although fluctuations in haplotype frequencies and the occurrence of a fourth haplotype in the historical population resulted in lower current levels of haplotype and nucleotide diversity. However, microsatellite markers revealed undiminished levels of nuclear diversity. No evidence for genetic structure was observed for the historical Spanish imperial eagle population, suggesting that the current pattern of structure is the direct consequence of population fragmentation. Temporal fluctuations in mitochondrial and microsatellite allelic frequencies were found between the historical and the current population as well as for each pairwise comparison between historical and current Centro and historical and current Parque Nacional de Doñana nuclei. Our results indicate an ancestral panmictic situation for the species that management policies should aim to restore. A historical analysis like the one taken here provides the baseline upon which the relative role of recent drift in shaping current genetic patterns in endangered species can be evaluated and this knowledge is used to guide conservation actions.