Ictalurus punctatus is native to the Americas and a superior freshwater fish cultured worldwide. However, acute mortalities often occur during aquaculture. In addition, the disadvantages of antibiotics have been highlighted, and a safe and effective substitute has become a hot topic. Thus, many studies have been published on the beneficial effect of probiotics, and most have targeted the microflora of the gastrointestinal tract, but few studies have investigated microflora of the gills and skin. To provide a theoretical basis to study gill, skin, stomach, foregut, and hindgut microorganisms and to screen probiotics from , we compared the microbial structures of the gills, skin, stomach, foregut, and hindgut from of different weights and explored the correlation with aquatic flora using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative-polymerase chain reaction (Q-PCR). All samples possessed rich bands, but the location, number, and color of the bands were quite different among samples. The mean numbers of DGGE bands detected in the organs from low to high were gills, skin, foregut, hindgut, and stomach. The Shannon diversity index and evenness and richness of bacteria in the skin, gills, stomach, foregut, and hindgut were 3.06, 0.94, and 23.00; 2.97, 0.92, and 19.67; 3.44, 0.93, and 31.33; 3.16, 0.93, and 23.50; and 3.30, 0.95, and 26.00 in low weight (LW) and 3.11, 0.99, and 23.83; 3.10, 0.96, and 22.33; 3.45, 0.94, and 31.50; 3.23, 0.94, and 25.05; and 3.28, 0.95, and 25.33 in high weight (HW) , respectively. The Shannon diversity index, evenness, and richness of gills and richness of the foregut from HW (I. punctatus than that in LW . Clustering and principal component analysis (PCA) distinguished LW from HW I. punctatus in the skin, gills, stomach, foregut, and hindgut were 0.82, 0.78, 0.78, 0.61, and 0.73, respectively, suggesting significant differences in the bacterial flora between LW and HW . In addition, the Shannon diversity index and evenness and richness of microflora in the aquatic environment were 3.45, 0.99, and 31.40, indicating slightly lower diversity than that of the stomach flora. The Q-PCR results showed that the dominant skin microflora were Enterobacteriaceae and , the dominant microflora in the water, gills, and stomach were Enterobacteriaceae, ; and the dominant microflora in the foregut and hindgut were Enterobacteriaceae, Flavobacterium (10^1.97) on the skin, Enterobacteriaceae (10^7.69) in the stomach, 3.83) in the foregut, and ), and ) in the hindgut were significantly higher in HW than those in LW <0.05). Moreover, the total numbers of bacteria in the skin, gills, stomach, and foregut (I. punctatus. These results reveal an increasing trend for the numbers of microflora in HW . The Q-PCR results were analyzed by PCA to identify the correlations between the microflora in all samples. The results demonstrated that LW and HW were distinguished by PCA1; the skin microflora was associated with that on gills, the foregut microflora was associated with that in the hindgut, and the stomach microflora was associated with that in the water. These results indicate the unique flora of the skin, gills, and gastrointestinal tract in and show that the structure and abundance of microflora are complex in LW and HW .