Amblyomma nodosum is a Neotropical tick species distributed from Mexico to Argentina, with adult individuals infesting different mammal species, including anteaters (Pilosa: Myrmecophagidae). Few reports in Colombia have recorded this species in departments such as Meta, Tolima and Valle del Cauca. In this paper we describe this species using taxonomic, morphometric and molecular methods after collecting individuals parasitizing collared anteaters (Tamandua tetradactyla) in the departments of Cundinamarca and Tolima. Adult specimens were identified based on current taxonomic keys and different morphometric variables were documented in nonengorged individuals. Also, DNA was extracted for PCR amplification and sequencing of 16S rDNA, COI and ITS2 genes. Phylogenetic trees were built. One hundred and thirty-four adult ticks were collected and identified as Amblyomma nodosum, based on morphological, morphometric, molecular and phylogenetic analyses. This is the first study to report the presence of this tick species in the department of Cundinamarca, using multiple identification approaches, thus expanding its geographical records in Colombia.