This study presents the first results on the evaluation and selection of polymorphic microsatellite markers for the genetic characterization of native chicken populations in southern Africa. Blood samples for DNA extraction were obtained from five chicken lines from South Africa (Koekoek, New Hampshire, Naked-Neck, Lebowa-Venda and Ovambo), and from one population each from Mozambique and Botswana. Twenty-three markers were found to be polymorphic in most of the lines studied. The mean number of alleles observed for all markers was 6.1, and varied from 2.3 (Koekoek) to 4.3 (Mozambique population). The lowest heterozygosity was found in the Koekoek line (31.4%), and the highest in the populations from Mozambique (60.7%) and Botswana (61.2%). It was concluded that this particular set of microsatellite markers can be of use as a measure of genetic variability in native chicken lines in southern Africa, and should be evaluated as diagnostic tools for breed identification.