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In this study microsatellite markers were applied to investigate the genetic diversity and population structure of the six local chicken lines kept in the “Fowls for Africa” program, for better clarification of parameters for breed differentiation and genetic conservation of this valuable resource. The lines included the Black Australorp, Potchefstroom Koekoek, New Hampshire, Ovambo, Lebova- Venda and a Naked Neck line. Unbiased estimates for heterozygosity ranged from 50% in the Potchefstroom Koekoek to as high as 65% in the Naked Neck chickens. FIS values varied from as low as 0.16 for the Black Australorp line to as high as 0.35 for the Ovambo chickens. The FST values indicated moderate to high genetic differentiation between the Naked Neck and New Hampshire (0.11); Ovambo and Naked Neck lines (0.12), and Naked Neck and Lebowa- Venda (0.14). A total of 13% of the total genetic variation observed was between the chicken lines and 87% within the lines, supporting moderate genetic differentiation. Population structure was assessed using STRUCTURE where the Black Australorp was genetically best defined. Although six clusters for the different populations could be distinguished, the other lines were not as clearly defined, with individual birds tending to share more than one cluster. Results support a broad classification of these lines and further investigation of unique alleles is recommended for conservation of the lines within the program.