The lack of a comparative approach makes it impossible to determine the main factors influencing colonization and evolution in plants. Here we conducted the first comparative study of a characteristic Mediterranean lineage (white-flowered Cistus) taking advantage of its well-known phylogenetic relationships. A two-scale approach was applied to address the hypothesis of higher levels of isolation in mountain than in lowland species. First, a time-calibrated phylogeny using plastid sequences of Cistaceae suggested that the origin of Cistus species postdated both the refilling of the Mediterranean Sea (5.59–5.33 Ma) and the onset of the Mediterranean climate (3.2 Ma). Two hundred and sixty-three additional, plastid sequences from 111 populations showed different numbers of haplotypes in C. laurifolius (7), C. monspeliensis (2) and C. salviifolius (7). Although haplotype sharing among disjunct populations was observed in all species, phylogeographic analyses revealed haplotype lineages exclusive to Europe or Africa only in the mountain species (C. laurifolius). Isolation by either geographical distance or sea barriers was not significantly supported for the lowland species (C. monspeliensis; C. ladanifer from a previous study). The same is true for the less habitat-specific species of the lineage (C. salviifolius). Comparative phylogeography of the Cistus species leads us to interpret a general pattern of active colonization surpassing Mediterranean barriers. In contrast, ecological conditions (precipitation, temperature, soils) appear to have determined the distribution of the Cistus species of Mediterranean mountains. This study further provides molecular evidence for multiple colonization patterns in the course of successful adaptation of Cistus species to Mediterranean habitats.