© 2015 Hildur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background The north-west coast of Spain was heavily contaminated by the Prestige oil spill, in 2002. Individuals who participated in the clean-up tasks showed increased chromosome damage two years after exposure. Long-term clinical implications of chromosome damage are still unknown. Objective To realize a follow-up genotoxic study to detect whether the chromosome damage persisted six years after exposure to the oil. Design Follow-up study. Setting Fishermen cooperatives in coastal villages. Participants Local fishermen who were highly exposed (n = 52) and non-exposed (n = 23) to oil seven years after the spill. Measurements Chromosome damage in circulating lymphocytes. Results Chromosome damage in exposed individuals persists six years after oil exposure, with a similar incidence than those previously detected four years before. A surprising increase in chromosome damage in non-exposed individual was found six years after Prestige spill vs. those detected two years after the exposure. Limitations The sample size and the possibility of some kind of selection bias should be considered. Genotoxic results cannot be extrapolated to the approximately 300,000 individuals who participated occasionally in clean-up tasks. Conclusion The persistence of chromosome damage detected in exposed individuals six years after oil exposure seems to indicate that the cells of the bone marrow are affected. A surprising increase in chromosome damage in non-exposed individuals detected in the follow-up study suggests an indirect exposition of these individuals to some oil compounds or to other toxic agents during the last four years. More long-term studies are needed to confirm the presence of chromosome damage in exposed and non-exposed fishermen due to the association between increased chromosomal damage and increased risk of cancer. Understanding and detecting chromosome damage is important for detecting cancer in its early stages. The present work is the first follow-up cytogenetic study carried out in lymphocytes to determine genotoxic damage evolution between two and six years after oil exposure in same individuals.