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Leaf-tailed geckos, genus Uroplatus, are one of the most prominent endemic reptile groups from Madagascar, but the species diversity and diversification of this taxonomic group are not completely understood. Here, we present a phylogenetic reconstruction of the small-sized Uroplatus which are included in the Uroplatus ebenaui group, focusing on the most widespread species of these geckos, Uroplatus phantasticus, which occupies a large mid-altitude distribution range in the eastern rainforests of Madagascar. Our phylogeny is based on DNA sequences of four mitochondrial genes (12S rRNA, 16S rRNA, COI and ND4), with a total of 2 312 base pairs. Partitioned Bayesian analysis confirmed U. malama from south-eastern Madagascar as the most basal representative of the group, followed by U. ebenaui from northern lowlands, and clades containing various undescribed candidate species as well as U. finiavana from Montagne d'Ambre in the north. Within our main targeted species U. phantasticus, the northernmost population from Zahamena (here considered as unconfirmed candidate species) forms the most basal lineage, while the southernmost populations studied from Marolambo, Ranomafana and Kianjavato form a nested clade, suggesting the species has expanded its range southwards undergoing repeated events of isolation and differentiation of lineages. Within the Ranomafana–Kianjavato area, we identified three different lineages, of which the two from Ranomafana are differentiated by 28 mutational steps in a fragment of the ND4 gene and occur on opposing banks of the Namorona River. This result suggests that this relatively small river currently limits dispersal and gene flow in these geckos. However, further downstream the Kianjavato population is more closely related to the population on the opposite side of the Namorona in Ranomafana, indicating that this genetic isolation is not absolute, and the river probably represents only a secondary barrier to gene flow in U. phantasticus. [ABSTRACT FROM AUTHOR]