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The small cavy Microcavia australis, a social and fossorial rodent, inhabits a large distribution range in South American arid zones. The species is versatile in coping with the seasonal and spatial variability typical of these environments through changes in morphology, physiology, and behavior. In order to explore whether phenotypic variations are related to the evolutionary history of the species, we analyzed the levels of genetic variability and divergence among four populations that differ in climate and habitat characteristics, two belonging to highlands and the other two from lowlands. We sequenced the mitochondrial control region and used the Inter Simple Sequence Repeats technique to study variability in the noncoding nuclear genome. Results from both genetic markers were consistent. Variability levels were high for all populations, and even higher for lowland ones. Pairwise genetic differentiation varied greatly, all comparisons being statistically significant except for the two highland populations. Seventeen haplotypes were detected which displayed three clear lineages: two corresponding to each lowland population and one to those in the highlands. Levels of genetic differentiation between population pairs varied widely. Haplotypes showed a mean sequence divergence of 1.4% between lowland populations and 0.2% between highland ones, whereas divergence was around 9% when populations from different altitudes were compared. Results from BEAST analysis support extant hypotheses suggesting that lowland forms are clearly older than the highland group. The deep genetic divergence between lineages poses the need to search for new evidence for properly defining the taxonomic status of divergent populations of M. australis. J. Exp. Zool. 315: 337-348, 2011. (C) 2011 Wiley-Liss, Inc.