Genetic quality of outbred stock has an important effect for the experimental results by using those animals, but methods and standardization of genetic detection for outbred stock are absent. In the present study, 6 microsatellite markers were screened for Wistar from Beijing and Spague-Darley (SD) from Shanghai outbred rats by fluorescence-based semi-automated genotyping method. Good polymorphisms were detected on all the 6 microsatellite loci, with 36 alleles found in the 2 stocks, 5-8 alleles each locus, and the polymorphic information content (PIC) ranged from 0.5892 (D11Mgh3) to 0.8019 (D6Mit1), with an average of 0.6881. 25 and 26 alleles were detected in the 6 loci, and averages of unbiased expected heterozygosity were 0.6260 and 0.6249 in Wistar and SD outbred rats, respectively. No significant differences of genetic diversity index were tested between populations. The Fst per locus was varied from 0.0461 to 0.4363, and the average Fst of all loci was 0.2069, which implied large genetic differentiation between populations. Nei's (1972) genetic distance and Nei's (1978) unbiased genetic distance measures between the 2 stocks were 1.2862 and 1.2726, respectively, which indicated the distant genetic relationship and low genetic identity between them. All loci in Wistar and 4 of 6 loci in SD outbred rats showed significant deviations from Hardy-Weinberg equilibrium (HWE), and all deviations were caused by deficiency of heterozygous individuals. From the results, there is abundant genetic variation in Wistar and SD outbred rats. Large genetic differentiation existed between these two outbred stocks, and each possessed distinct genetic characteristic. Deviation from HWE seems a frequent problem in the 2 outbred stocks. This genetic research on outbred rats should assist in developing genetic monitoring methods and standardization of the outbred rats.