Wild boars are natural hosts for African swine fever (ASF). The ASF virus (ASFV) can persist for long periods in the environment, such as in ticks and contaminated products, which may be sources of infection for wild boar populations. African swine fever was eradicated in domestic pig populations in Spain in 1995, after 35years of significant effort. To determine whether ASFV can persist in wild boar hosts after it has been eradicated from domestic pigs and to study the role of wild boar in helping ASFV persist in the environment, we checked for the presence of ASFV in wild boars in Doñana National Park, one of the largest natural habitats of wild boar in Spain and one of the last areas where ASF was endemic prior its eradication. Samples from 158 animals collected between 2006 and 2010 were analysed using serological and nucleic acid-based diagnostic techniques recommended by the World Organization for Animal Health (OIE). None of the samples was found to be positive. These results confirm the absence of disease in wildlife in what was once one of the areas most affected by ASF in Spain, and they suggest that wild boars play a limited role in ASFV persistence. These results confirm that ASFV cannot persist in isolated wild boar populations for long periods of time without the interaction of other factors such as re-infection by contact with domestic pigs or by feeding on contaminated swill. ; This work is a contribution to JCCM PPIC10-0226-0243 and was partly funded by ASFRISK project (EC, FP7-KBBE-2007-1, Project no. 211691). possible. Lina Murholds a scholarship of the FPU Program (Ministry of Education and Science, Spain). ; Peer Reviewed