Amblyomma multipunctum is a poorly known Neotropical tick (Acari, Ixodidae) described in 1899. It has been found associated with large mammals such as tapirs (Tapirus), and the Andean bear (Tremarctos ornatus). Males, females and nymphs have been described; however, the larvae of the species remain unknown. Valid records for A. multipunctum are few and limited to three countries in northwestern South America: Colombia, Ecuador, and Venezuela. In the present work, two partially engorged nymphs of A. multipunctum parasitizing the northern pudu, Pudu (Pudella) mephistophiles, were collected near to the Puracé National Natural Park, Department of Cauca, Colombia. Ticks were identified by morphology, with diagnostic characters such as scutum with multiple punctuations, two separate spurs in coxa I (the external being longer than the internal), a small triangular spur in coxae II–IV, spiracular plate triangular with rounded angles and a vestigial prolongation in article I of the palps. In addition, the taxonomical identity was validated using molecular evidence by PCR amplification of a fragment of the 16S rDNA gene, showing an identity percentage of 100% comparing with sequences in GenBank for A. multipunctum. This study reports the first association of A. multipunctum with P. mephistophiles, the extension of geographical distribution in Colombia, extending its elevational range by 644 m in the country, and contributing to the knowledge of the intraspecific variation of the tick nymph.