A well-documented consequence of repeated global ice ages is the negative relationship between latitude and intraspecific genetic diversity. However, little is known about additional effects of such major climatic events on population genetic structure. Here we studied the phylogeographic structure of five lycaenid butterfly species with varied ecological adaptations, sampled across a latitudinal gradient in the Holarctic region. We found a positive correlation between latitude and substitution rate of mitochondrial DNA sequences in all species investigated. We propose that this result is the signal of increased genetic drift and founder effects during post-glacial recolonization of northern populations. Given that phylogenetic branch length is the result of a cumulative process over evolutionary time, we hypothesize that a latitudinal layering of populations has generally been maintained during repeated cycles of glaciation, possibly due to a neutral spatial effect and/or local adaptive advantage. This trans-glacial latitudinal layering could be viewed as a particular case of the more general phenomenon of intraspecific structuring that is created and maintained in a fluctuating environmental gradient.