Microsatellite DNA fingerprinting technology was used to investigating the genetic variation and germplasm identification within three tilapias of Oreochromis aureus，O. niloticus and red O. mossambicus. 82 pairs of primers which could give stable and polymorphic amplification profiles were screened out from 103 microsatellite loci and used to analyze the genetic structure of three varieties. Totally, 605 alleles of microsatellite loci were obtained and the average number of alleles was 7.37. Additionally, microsatellite DNA fingerprinting database of three tilapias was established based on the results of alleles of microsatellite loci by Popgen32 software package and illustrative patterns were constructed by EXCEL. The results showed that, the mean value of observed heterozygosity of O. aureus, O. niloticus and O. mossambicus was 0.179 6, 0.530 1, 0.312 2, the mean value of expected heterozygosity was 0.203 2, 0.678 6, 0.291 3, and the polymorphism information content (PIC) was 0.075 4, 0.608 3, 0.152 8, respectively. It indicated that, the genetic diversity of O. niloticus was the highest and O. aureus was the lowest. Then 16 pairs of primers that can distinguish one variety tilapia from the others were screened out as the core primers for tilapia germ plasm identification. The amplified data from 16 pairs of special primers were transformed to the application platform of digital microsatellite DNA fingerprinting, which could be used to identify tilapia species. This result could provide a technique for solving the problem of germplasm farrago for tilapia.