Molecular markers are a useful tool for evaluating genetic diversity of chicken genetic resources. Seven chicken lines derived from the Plymouth Rock breed were genotyped using 40 microsatellite markers to quantify genetic differentiation and assess conservation priorities for the lines. Genetic differentiation between pairs of the lines (pairwise FST) ranged from 0.201 to 0.422. A neighbor-joining tree of individuals, based on the proportion of shared alleles, formed clearly defined clusters corresponding to the origins of the lines. In Bayesian model-based clustering, most individuals were clearly assigned to single clusters according to line origin and showed no admixture. These results indicated that a substantial degree of genetic differentiation exists among the lines. To decide priorities for conservation, the contribution of each line to the genetic diversity was estimated. The result indicated that a loss of 4 of the 7 lines would lead to a loss from 1.14 to 3.44% of total genetic diversity. The most preferred line for conservation purposes was identified based on multilocus microsatellite analysis. Our results confirmed that characterization by means of molecular markers is helpful for establishing a plan for conservation of chicken genetic resources.