Hard ticks of the genus Ixodes are one of the most diverse groups in the Neotropical region, but some species are known only from historical records. In Colombia and Venezuela, Ixodes montoyanus is known from records obtained more than 50 years ago. In this study, we performed morphological and molecular evaluations of immature and adult stages of I. montoyanus collected from Pudu (Pudella) mephistophiles, in the municipality of Puracé, Cauca, in the Andean region of Colombia. The results include the first morphological evaluations of larvae, nymphs, females and males of I. montoyanus, confirming each of these stages with their respective sequences of a fragment of the mitochondrial 16S rRNA gene, and nymphs and females with a fragment of the mitochondrial cox1 gene. These results represent the first molecular information of I. montoyanus from Colombia, allowing differentiation of this species from others within Ixodes distributed in the country. The data generated consolidate valuable information for the genus Ixodes, which plays an important role in pathogen transmission. The information is also key to addressing the controversy regarding the identification, validity and distribution of various species within the genus.