Of the 44 species within the genus Dermacentor (Acari: Ixodidae) worldwide, nine are found in the Neotropics and four are endemic to this region. In Colombia, located in the northwestern corner of South America, there are two species of the genus: Dermacentor imitans restricted to the trans-Andean Pacific region, and Dermacentor nitens widely distributed in the country. Elsewhere, D. nitens is found in both the Nearctic and Neotropics, from southern USA to northern Argentina. In the Neotropics, D. nitens is widely distributed including records in Caribbean islands. Molecular analyses of D. nitens collected in the Amazon region of Colombia have shown genetic differences with specimens from other localities of the country, although morphological differences have not been explored. We performed morphological and molecular comparisons of individuals from two natural regions of Colombia: the Orinoquia, and the Amazon. The morphological and morphometric results show that females and males are grouped in two morphotypes: morphotype I from the Orinoquia region, and morphotype II from Leticia in the Amazon region. The molecular analyses supported the morphological results and showed the formation of two well-differentiated sister clades within D. nitens, with divergences between both morphotypes of 3–4% and 5–6% for the 16S rRNA and COI genes, respectively. Species delimitation analyzes and the Poisson Tree Processes model tentatively suggest the presence of two species within the sequences associated with D. nitens in Colombia. The first represented by the sequences from the Orinoquia region evaluated here, together with sequences from other regions of Colombia and other Neotropical countries such as Brazil, Panama, and French Guiana. The second is represented by the sequences of individuals from Leticia, in the Amazon region of Colombia. The results highlight that D. nitens might represent a species complex, but further sampling, experimental crosses, and studies at the level of complete genomes should be performed to obtain additional evidence for species recognition.