We combined tools of phylogeography, population genetics and biogeographical interpretation to analyse a group of phylogenetically independent lineages (animals and plants) that coexist within the same geographical region, yet under markedly different environments, in order to identify generalized barriers for gene flow. We tested the hypothesis that major geographic features have produced a concordant genetic structure in phylogenetically independent lineages. A rigorous bibliographic search was performed, selecting available molecular information from six taxa occupying distinct southern biomes of South America: Yungas, Prepuna, Puna and northern Monte. We estimated within-population genetic diversity, the genetic structure and haplotype phylogenies to assemble distribution maps of genetic barriers for each species. We found a strong association between genetic variation and latitudinal distribution of populations. We detected a major barrier for six taxa at 27°S latitude and a second one for a group of three species at 25–26°S. Two alternative non-exclusive hypotheses – geology and/or climate – explain concordant genetic barriers in divergent lineages. We suggest that the term ‘biogeographically significant units’ portrays a group of populations of phylogenetically unrelated taxa that inhabit the same geographic region that have been similarly impacted by major physical events, which can be used to identify priority areas in landscape conservation.