We collected fishes at the 12,873 sites in stream order (1~7) from 2008 to 2016. In the results, two populations (Zacco platypus and Zacco koreanus) represented distributional differences in the stream order gradient, and correlation analysis showed that the two populations had a potential competitive relationship. The ecological characteristics of the fish except for the Z. platypus and the Z. koreanus, according to the gradient of stream order showed a pronounced gradient in the species such as intermediate species, sensitive species, carnivores, herbivores and omnivores. The two populations showed a high correlation between intermediate species, sensitive species of the tolerance guild and omnivores, insectivores of the trophic guild. Fish assessment index (FAI) was negative relation with stream order. According to the stream order gradient, the two populations showed different correlations with FAI.