Abstract: DNA fingerprinting offers great potential in aquaculture and in fisheries as a tool for identification of individuals, construction of pedigrees and population genetic analysis. Microsatellites, also called simple sequence repeats (SSR), are widely dispersed along and among chromosomes, and each locus is characterized by a known DNA sequence. Due to a tendency for hypervariability and because only small amounts of tissue are required for typing, microsatellites have supplanted allozymes in recent years as the genetic markers of choice for many biological problems including parentage assessment, genomic mapping and establishment DNA fingerprinting. Largemouth bass (Micropterus salmoides) was introduced from Taiwan to Guangdong province of China in 1983, because of delicious taste, fast growth, and wide temperature tolerance, it has been bred in many region of China as a main cultured fish species with annual production of 100,000 tons. The largemouth bass consists of two subspecies, the Florida largemouth bass (M. s. floridanus), which is native to peninsular Florida, and the northern largemouth bass (M. s. salmoides), whose native range extends throughout the central and eastern U.S. as well as northeastern Mexico and southeastern Canada. Research shows that largemouth bass cultured in china attribute to the northern largemouth bass (M. s. salmoides). In this paper, the microsatellite DNA fingerprinting of four largemouth bass populations was established, including cultured stock of M. salmoides in China (CH), M. s. floridanus was introduced in 2009 (FL-09), M. s. floridanus was introduced in 2010 (FL-10) and M. s. salmoides was introduced in 2010 (NT-10), and the genetic structure of them was analyzed by 43 microsatellite markers. The results showed that a total of 246 alleles were obtained from the four largemouth bass populations, and the alleles ranged from 2-13 in each locus. The mean value of number on alleles (A) and the mean value of expected heterozygosity (He) were calculated by popgene 3.2 software. The result was that the mean value of number on alleles (A) of CH, FL-09, FL-10 and NT-10 was 2.58, 3.74, 3.70 and 4.21, the mean value of expected heterozygosity (He) was 0.4549, 0.4896, 0.5010 and 0.6138, and the mean value of polymorphism information content (PIC) was 0.3786, 0.4443, 0.4566 and 0.5546, respectively. It indicated that the genetic diversity of cultured stock on largemouth bass in China was lower than that of introduced largemouth bass populations. Using Unweighted pair-group method with arithmetic means method (UPGMA) based on their genetic distances, the cluster analysis in four populations showed that FL-09 and FL-10 were grouped together, genetic distance was 0.0506; The NT-10 and CH were grouped together, genetic distance was 0.4244. The results showed that the FL-09 and FL-10 of M. s. floridanus belonged to same population, but NT-10 and CH of M. s. salmoides came from different populations, and even different river system. Then five pairs of specific microsatellite markers (JZL114, MiSaTPW11, Lma120, Mdo6 and Msal21) were screened out from microsatellite DNA fingerprinting which could be used to identify FL, NT-10 and CH populations. Especially microsatellite markers (MiSaTPW11 and Msal21) combination could completely identify FL, NT-10 and CH populations. The amplified data from five pairs of special markers were transformed to the application platform of digital microsatellite DNA fingerprinting, which could be used to identify largemouth bass species and hybrid identification. These results could provide a theoretical basis for germplasm conservation, species identification and breeding of largemouth bass in China.