The aims of the current study were to assess genetic diversity, conduct genetic characterization, and evaluate usefulness of an individual assignment test for 12 commercial chicken lines using 40 microsatellite markers. A total of 268 distinct alleles were observed across the 12 lines, and 42 of the 268 alleles (15.7%) were unique to only 1 line. Mean observed heterozygosity within a line ranged from 0.295 to 0.664, and the highest value was obtained from 1 of the White Plymouth Rock lines. Significant deviations from the Hardy-Weinberg equilibrium were observed at several locus-line combinations, showing excess of heterozygotes in many cases. As a whole, genetic differences among the lines estimated by the fixation index were high at 29.8%, whereas higher genetic similarity was observed among White Leghorn lines despite their different breeding histories. Assignment test could correctly allocate individuals at the line level to their origins, with a high accuracy (96.6%). Individual-based genetic characterization would be a usable step to conserve chicken genetic resources. Here, guidelines for future breeding and management of these lines by the poultry industry are provided.