Knowledge of genetic diversity and relationship among domestic animals is important for sustainable strategies and conservation plans evaluating genetic diversity within and across breeds. Due to the lack of studies focusing on the genetic relationship between the European and South American creole populations, we performed comprehensive genetic analyses of nine sheep breeds raised in Spain compared with Araucana creole sheep. The Araucana creole sheep is an autochthonous breed raised in the south of Chile and represents an important zoogenetic resource for the local economy. The aim of this research was to characterize the genetic variability and structure based on individual genotypes. A total of 179 individuals were analyzed (Ile de France, IF n = 18, Merino Espanol, ME n = 18, Merino de Grazalema, MG n = 16, Merino Negro, MN n = 18, Montesina, MO n = 19, Merino Precoz, MP n = 18, Segurelia, SE n = 17, Churra Lebrijana, CL n = 14, Fleischschaf, FL n = 13, Araucana, AR n = 28) by using 19 microsatellite markers. In total, 281 alleles were observed, and the value of average polymorphic information content was 0.81. The estimated diversity parameters indicated that populations retained high levels of genetic diversity (0.69 0.06) and revealed the existence of genetic differentiation among the groups (FsT = 10.1%). We found that the highest inbreeding coefficient was in the MP (FIs = 0.121). The study also proved the short genetic distance between ME and MG, whereas MO and MP were the most distant populations. Finally, AR presented a short genetic distance with the SE breed. The STRUCTURE analyses revealed that the populations have a particular genetic background and presented different levels of admixture. The most appropriate cluster number was K = 7. This work is the first report to confirm historical information about the genetic origin of the domestic creole population in Chile and its relationship with European breeds. Overall, the population parameters reported here provide useful information for establishing support in terms of decision-making about their conservation.