In several insects, there appears to be a high fitness cost associated with insecticide resistance genes during the overwintering period. In order to understand when and how this cost operates, all mosquitoes entering a natural cave for overwintering were regularly sampled, and their resistance genes at two loci (Ester and Ace.1) were individually identified. During the main period of entry (October and November), susceptible mosquitoes were first observed, followed by resistant ones, this trend being similar for both loci. This observation is best explained by a migration phenomenon, northern and more susceptible populations starting to overwinter first, followed by southern and more resistant ones. During the remaining part of the winter (December–March), mosquitoes entering the cave were still caught and they probably corresponded to individuals leaving a former overwintering site in search of a more suitable one. A lower overall frequency of resistant phenotypes was found in the second part of the winter at Ester, suggesting that a large fitness cost (42%) had operated. A decreasing frequency of resistant phenotypes was also found at Ace.1, indicating a large survival cost of resistant mosquitoes (7% for the homozygote Ace.1R) in their former overwintering site. These results are discussed in the light of the local evolution of these resistance genes in southern France.