After a morphological analysis of several tarantula specimens of the genus Eupalaestrus, including recently collected specimens from previously unrecorded localities, we herein describe two new species. They differ from congeners by the presence of a large area with spiniform setae between the coxae IV. They differ from each other in general size and in the thickening of the tibia and metatarsus IV. Eupalaestrus crassimetatarsis sp. nov. is distributed in the Brazilian States of Paraná, Rio Grande do Sul and Santa Catarina, and in Misiones Province in Argentina. Eupalaestrus roccoi sp. nov. is distributed in the Brazilian state of Rio Grande do Sul. These natural grasslands are under threat from rapidly encroaching arable and livestock farming.