Abstract: Based on the allele frequencies of 29 microsatellite loci, the accuracy of pair wise genetic distance among 4 Chinese native chickens was analyzed at 5, 10, 15, 20 and 25 microsatellite loci levels. The results indicated that the accuracy of genetic distance estimation increased along with the numbers of the loci increase. The result of t test was that 25 microsatellite loci had significant difference with 5, 10, 15 and 20 loci levels. It could be concluded that more possible microsatellite loci should be used when the experiment was designed to analyze genetic relationship between different varieties.