Abstract: Loaches is one group of small freshwater fish dwelling in slow to moderately swift water, widely distributes in rivers and lakes. Poyang Lake, the biggest fresh water lake in China, has a rich biodiversity in fishes. The loaches of the Poyang Lake can be divided into three kinds based on skin spot: the big-spot loaches, the small-spot loaches and the non-spot loaches. Preliminary research indicated that the biological character of its skin spot was stable, which belongs to quantitative traits loci (QTL). In this study, with the expectation, to provide the evidence of theory in well-bred loach selection, isozyme electrophoresis, RAPD and mtDNA Cyt b gene sequencing were applied to investigate the genetic diversity of the three different kinds of loaches. Using discontinued polyacrylamide gel electrophoresis to analysis the isozymes, results showed that there were some genetic differences among the three kinds loaches based on LDH, MDH and EST isozymes. These differences were not only among different tissues, e.g. LDH4 only existed in gonad tissue just as m-MDH-A in muscle tissue and EST-2 in liver tissue, but also among the three kind loaches, e.g. m-MDH-B only existed in muscle tissue of the big-spot loach but not in other two kinds loaches, cLDH was only existed in the liver tissue of the small-spot and non-spot loached not find in the big-spot loach. RAPD technique, as an important tool for genetic study, was widely used in identification of species. 6 random primers, with more stable and reproducible characteristics, were selected from 35 random primers in RAPD to evaluate the genetic diversity. 169 distinguishable DNA fragments were amplified in all, varying in length from 200 to 1800 bp, 101 of them (59.56%) were polymorphic. Each primer could amplify DNA fragments varied from 8 to 11, 9.39 fragments per primer, including 5.61 polymorphic fragments. The shannon’s index was 0.3750 in the big-spot loaches, 0.2706 in small-spot and 0.2656 in none- spot loaches. So the genetic similarity index by the result of RAPD in the three kind loach were very high from the nuclear DNA. The Cyt b gene, 1140bp sequences, was obtained by PCR amplification in the three kind loaches. Four haplotypes were identified from 28 samples with 113 variation site, one was the mutual haplotype and others were endemic haplotypes. The endemic haplotype which presented only in big-spot loach had 91 variation sites compared to the mutual haplotype, but the endemic haplotype in small-spot and non-spot loach only had 20 and 24 variation sites. With Myxocyprinus asiaticus as outgroup, the phylogenetic trees were analyzed using the neighbor joining (NJ) method and the maximum parsimony (MP) in MEGA 4.0. The result showed that the haplotype of the big-spot loach were clustered into another branch with high confidence level, it also indicated that there were a certain genetic differences among the three kinds loached based on mitochondrial cytochrome b gene sequences. The genetic diversity of the three kind loaches in Poyang Lake, based on the results of isozymes, nuclear DNA (RAPD analysis) and mtDNA (Cyt b gene analysis), was abundant. Results also indicated that the loach in Poyang Lake might have population heterozygosis.