Historically, the identity and distribution of some Bothriurus species from Patagonia have not been clear. The previous concepts of the Bothriurus patagonicus species group may comprise more than one species. Two new species, Bothriurus huincul sp. n. and B. sanctaecrucis sp. n., are described, and additional morphological and distributional data of B. patagonicus, and B. burmeisteri are provided. An identification key and a map with all the records of Patagonian Bothriurus species are provided. Phylogenetic and biogeographic aspects of Patagonian Bothriurus, reflecting ancient connections of Patagonia with the center and north of Chile, are discussed.