The objective of this study was to determine genetic variation of five Korean native chicken lines using 30 microsatellite(MS) markers, which were previously recommended by ISAG(International Society for Animal Genetics). The initial study indicated that two microsatellite markers, MCW0284 and LEI0192, were not amplified in these lines and excluded for further analysis. Twenty eight microsatellite markers were investigated in 83 birds from five Korean native chicken lines. The identified mean number of alleles was 4.57. Also, the expected, observed heterozygosity(He, Ho) and polymorphism information content(PIC) values were estimated in these markers and they ranged from 0.31~0.868, 0.145~0.699, and 0.268~0.847, respectively. The results were used for the discrimination of five chicken lines using genetic distance values and also neighbor-joining phylogenetic tree was constructed. Based on the He and PIC values, eighteen markers are enough for the discrimination of these Korean native chicken lines for the expected probability of identity values among genotypes of random individuals(PI), random half sibs(PIhalf-sibs) and random sibs(PIsibs). Taken together, these results will help the decision of conservation strategies and establishment of traceability system in this native chicken breed. Also, the use of ISAG-recommended microsatellite markers may indicate that the global comparison with other chicken breeds is possible.