The Mapuche fowl is an autochthonous breed raised in Chile and represents an important zoogenetic resource for the local economy. This study aimed at investigating the genetic diversity, relationship and population structure of 96 local Chilean chickens derived from 3 ecotype of Mapuche fowl (Kollonka, Ketro, and Kollonka de aretes), 2 ecotype Chilean (Trintre, Cogote pelado) and 2 breeds (Light Brahma and Barred Plymouth Rock) using 12 microsatellite markers. In total, 113 alleles were detected in all populations, with a mean of 7.6 alleles per population. In all population chicken breeds, the observed and expected heterozygosity ranged from 0.91 to 0.98 and from 0.69 to 0.79. Furthermore, all populations showed significant deviations from Hardy-Weinberg expectations. Across each population, the global heterozygosity deficit (FIT) was -0.174, population differentiation index (FST) was 0.073, and the global inbreeding of individuals within breed (FIS) was -0.267. The phylogenetic relationships of chickens were examined using neighbor-joining trees constructed at the level of population. The highest Nei's standard genetic distance value of 0.559 was observed between Barred Plymouth Rock and Light Brahma, whereas the minimum value (0.099) was found between Kollonka and Trintre. The neighbor-joining tree constructed at population level revealed 2 main clusters, with Light Brahma, Barred Plymouth Rock, Ketro and Kollonka de aretes in 1 cluster, and Kollonka, Trintre and Cogote pelado breeds in the second cluster. Based on the results of the STRUCTURE analysis, the most likely number of clustering of the population evaluated was at K=3, with Light Brahma and Barred Plymouth Rock breeds forming their own distinct clusters, while Kollonka, Ketro, Kollonka de aretes, Trintre and Cogote pelado breeds clustered together. This study represents the first report of genetic diversity in these populations in Chile. These results can be used as baseline genetic information for genetic conservation program, for instance, to control inbreeding and to implement further genetic studies in local Chilean chickens.