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Simple Summary Creole animals are an important genetic resource due to their adaptation to adverse conditions, however, many of them have disappeared or have been reduced in number; therefore, their identification and preservation should be promoted. In the Mixteca zone of Oaxaca, México, there is a group of creole sheep with physical differences with respect to the other known breeds. The objective of the present study was to determine the degree of differentiation among the individuals of the population of creole sheep and the similarities with other breeds, as well as to measure their level of conservation and consanguinity. It was found that there is a group of sheep which is different from the presently known breeds and that they do not present high degree of consanguinity, and thus may be considered a new breed. Therefore, it is proposed that they are identified as the Chocholteca Creole breed, in honor of the ethnic group which inhabits this region. The present study is very important because it discovers a new genotype of sheep, which amplifies the genetic diversity. Therefore, further studies are needed, given that they are a potential alternative of meat and wool. Abstract Creole sheep in México have undergone crossbreeding, provoking the loss of genetic variability. The objective of the present study is to determine the intra-racial genetic diversity, the genetic relationship with other genotypes, and the populational substructure of the Oaxacan Creole sheep. Twenty-nine blood samples were obtained of Creole sheep of the Oaxaca Mixteca region in México. A genetic analysis was made with 41 microsatellites recommended for studies of genetic diversity in sheep. An analysis was made of genetic diversity, populational structure, and genetic distance with 27 other sheep populations. The study found 205 alleles with a range of 2 to 9 by locus and an effective number of 3.33. The intra-racial analysis showed a moderate genetic diversity with values of expected heterozygosity of 0.686 and observed of 0.756, a mean polymorphic information content of 0.609, and a mean coefficient of consanguinity of −0.002. In interracial genetic diversity for the coefficients of consanguinity, the values were FIS = 0.0774, FIT = 0.16993, and FST = 0.10028, showing an elevated genetic distance with other creole breeds, but close to Argentine Creole, to another Creole of México and the Spanish Merino. Its genetic structure showed that it does not have any populational subdivision nor mixes with the others analyzed. It is concluded that it is a distinct and isolated population and is proposed as the creole breed “Chocholteca” for its conservation.