A number of Korean Chicken breeds were registered in Domestic Animal Diversity Information System (DAD-IS, http://dad.fao.org/) of the Food and Agriculture Organization (FAO). Evaluation of genetic diversity and relationship of local breeds is an important factor towards the identification of unique and valuable genetic resources. Therefore, this study aimed to analysis the genetic diversity and relationship of 22 Korean Chicken breeds using 12 microsatellite (MS) markers. The mean number of alleles for each variety was 5.52, ranging from a 3.75 (Leghorn F; NF) to a 7.0 (Ross). The most diverse breed was the Hanhyup3 (HCC), which had the highest expected heterozygosity (HExp) (0.754) and polymorphic information content (PIC) (0.711). The NF was the least diverse population, having the lowest HExp (0.467) and PIC (0.413). As a result of the principal coordinates analysis (PCoA) and factorial correspondence analysis (FCA) confirmed that Hy-line Brown (HL) and Lohmann Brown (LO) are very close to each other and that Leghorn and Rhode Island Red (RIR) are clearly distinguished from other groups. Thus, the reliability and power of identification using 12 types of MS markers were improved, and the genetic diversity and probability of individual discrimination were confirmed through statistical analysis. This study is expected to be used as basic data for the identification of Korean chicken breeds, and our results indicated that these multiplex PCR marker sets will have considerable applications in population genetic structure analysis.