A new subspecies of the Tropidurus spinulosus group is recognized and described as Tropidurus spinulosus guarani. Its distribution area corresponds to the wet mesic region from central Paraguayan districts east of Asunción and Paraguay River. Morphological characters and peculiar features of the new taxon are analysed and discussed in comparison with Cope’s nominal form. Populations of Tropidurus spinulosus spinulosus from the xerophytic Chacoan region of Argentina were used. Morphological differences between the uniform arboreal populations from northern and central Chacoan flats and the southernmost saxicolous populations from the Córdoba embossments are also reported and discussed.