In this study, indigenous chicken populations representing seven different areas of northwest Ethiopia were studied using microsatellite markers to determine genetic diversity and variation. Three local lines of South African chicken and two commercial chicken strains were included for comparison. The Ethiopian chicken population Gassay/Farta had the highest number of alleles per locus (10) for microsatellite marker MCW 158. MCW 154 was the most polymorphic marker across all populations with an average of seven different alleles. A high genetic diversity was observed in overall loci for all populations with heterozygosity (Ho) value of 0.77. The highest heterozygosity (0.93) across all markers was observed in the Mecha chicken population, while the lowest heterozygosity across all loci (0.66) was observed in the White Leghorn breed. The RIR commercial chicken breed showed higher genetic distance (lower genetic similarity) with the Ethiopian chicken populations than the South African fowls. This indicates that the Ethiopian indigenous chicken populations are still not highly diluted by the RIR chicken breed either through the extension program or through the regional poultry breeding and multiplication institutes. Based on the phylogenetic tree result, it is concluded that the clustering of the chicken populations in the present study are in accordance with the origin and marketing systems of these native chickens, which indicates that the microsatellite markers used in this study were suitable for the measurement of the genetic biodiversity and relationship of Ethiopian chicken populations. These results can therefore serve as an initial step to plan the characterization and conservation of native chickens in Amhara region, Ethiopia.