Simple Summary Creole sheep were introduced in South America by Spanish colonizers by the end of the fifteenth century. However, the exact dynamics of their formation remains uncertain. There is no consensus on the arrival of hair sheep of African origin, which may have reached the Caribbean coasts at different periods, compared to wool sheep that arrived from the Iberian Peninsula. Although bovines are the most important species for animal-origin food production in South America, sheep represent a strategic genetic resource for populations living in marginal areas with scarce economic resources. This study sheds light on the genetic structure of Colombian Creole sheep on the basis of mitochondrial DNA analysis. The main results indicate a common genetic layer originating from the first sheep that arrived during the European colonization, followed by the introduction of sheep of African origin. Abstract The genetic origins and diversity of Creole sheep from five regions of Colombia were investigated based on mitochondrial DNA (mtDNA) variations across 89 sequences from five breeds: one wool Creole sheep (CL) and four hair Creole sheep, including Ethiopian (OPCE), Sudan (OPCS), Pelibuey (OPCP) and Wayúu (OPCW). A global comparison was done using 62 haplotypes from Iberian, African, Indian, Caribbean, Mexican, Caucasian and European sheep based on sequences retrieved from GenBank. This study aimed to identify the maternal origin of Colombian Creole sheep and their genetic relationships at a global level. The results showed 31 different haplotypes from Colombian Creole sheep, which can be assigned to maternal lineage B, the most common lineage found in European sheep breeds and the only one found in several Iberian breed (e.g., Churra, Spanish Merino) that most likely participated in the Creole formation. Additional analyses showed that wool and hair sheep retained a broad genetic identity despite being geographically separated. The global-level phylogenetic analysis revealed that Colombian Creole sheep belong to a distinct and defined genetic lineage that is likely the result of a founder effect with ecotypes of Iberian descent and the subsequent introduction of foreign breeds. This is consistent with historical reports on the presence of sheep in South America and, particularly, Colombia.