Transition zones between bioregions can sustain the coexistence of species with different biogeographic affinities through ecological niche segregation at a finer scale. Reptiles can serve as a good model group to evaluate the mechanisms behind such coexistence patterns especially due to their thermal sensibility to microhabitat structure. We examined whether reptile species spatially segregate in their microhabitat use and whether this segregation reflects the biogeographic affinities of species. Reptiles were surveyed across a 1.5-km transect located in the Peneda-Gerês National Park. We used 478 GPS observations of nine sympatric non-Mediterranean and Mediterranean species collected during field surveys in 2012 and 2013. Each observation was assigned to a microhabitat-openness measurement. Differences in microhabitat selection between Mediterranean and non-Mediterranean species were studied with factorial ANOVA analyses. The similarity in microhabitat selection between pairs of species was compared with th similarity in their Iberian distributions by a Partial Mantel test checking for the genetic distances between species pairs in a phylogenetic framework. Comparing a random selection of points along transect, we found that non-Mediterranean species used more closed microhabitats than Mediterranean species. No differences in this pattern were found between snakes and lizards. The Mantel test showed that microhabitat selection was correlated with the Iberian geographic distributional patterns of species. The complex vegetation structure of this area is most likely responsible for the availability of different microhabitats, which promotes high reptile species richness and governs their sympatric coexistence at a finer scale.