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Six mouse opossum species are currently recognized for the genus Thylamys in South America, of which we hypothesized the evolutionary relationships for those located in the Andean Altiplano and canyons of the Region I of Chile, as well as for those located south of the Atacama Desert down to 37degrees S. To that goal we sequenced the cytochrome b mitochondrial gene and data were analyzed using different phylogenetic criteria such as parsimony, distance and likelihood available in the program PAUP 4.0. The different phylogenetic approaches agreed in recovering two well defined clades: one constituted by Thylamys pallidior that included specimens from the coast and the "pre-cordillera" of the Region I of Chile, and the other clade constituted by Thylamys elegans that included populations located south to the Atacama Desert. Therefore, we demonstrate that populations located in the canyons and andean areas of northern Chile belong to T. pallidior, while those located south to the Atacama desert are recognized as T. elegans, results that confirm the occurrence of two thylamyine species in Chile.