Simple SummaryMitochondrial DNA (mtDNA) analysis is a tool in assessing the maternal origin, phylogeny, and population structure of domestic animals. The Ecuadorian Pillareño Creole pig is a creole breed that comes from the descendants of Iberian pig introduced to Ecuador by the Spanish conquerors. This creole population represents important reservoirs of genetic diversity that are very essential to preserve; however, the introduction of new breeds of pigs has displaced this creole pig from Ecuador to the background. The objective of the mitochondrial DNA analysis was to identify the Ecuadorian Pillareño Creole pig populations, their origins, and their maternal lines. For this study, DNA samples from 34 Ecuadorian Pillareño Creole pigs were used, with the animals belonging to seven rural regions of Ecuador. The haplogroup network suggested that the Pillareño creole pig population can be grouped into a single haplotype and that it belongs to the European pig clades. The genetic relationship between the Ecuadorian Pillareño Creole and the European pigs, particularly the Iberian pigs from Spain, can be used to establish of an official breeding program for the conservation and valuation of these creole populations, with this genetic mitochondrial analysis potentially providing a better approach for the rescue of the Ecuadorian Pillareño Creole pig populations.Domestic pig breeds reached America on the second Columbus trip; from this date, Iberian pig genetic resources were disseminated throughout the continent, forming diverse creole breeds. These Ecuadorian Creole pigs are important for food production but have been genetically eroded since the introduction of transboundary breeds. In this study, we sought to characterize this erosion more thoroughly through mitochondrial DNA D-Loop analysis of Ecuadorian Pillareño Creole pigs from seven regions of Ecuador. To allow comparison, we also included in our analysis sequences from wild species, commercial lines, and domestic pigs, which were obtained from the NCBI GenBank database. Creole pigs’ population showed overall moderate Hd values and low π values, and a negative value of Tajima’s D was observed. The greatest differentiation from the Ecuadorian Pillareño Creole pigs was observed between Asian wild and Asian domestic pigs. The haplotype analysis revealed three different phylogenetic clades (A, E I, and E II) and 65 haplotypes. Ecuadorian Creole populations were grouped into nine haplotypes for Clade E I and E II, which have not previously been reported for Creole Pillareño populations. Our analysis indicates that in the establishment of Creole Pillareño pigs, individuals most likely separated from the Asian pig population and appear to be genetically influenced by European and Iberian populations raised in Spain.