Stream development can generate environmental changes that impact fish communities. In temperate streams, the distribution of fish species is associated with environmental gradients. To analyze the relevant factors, large-scale exploration is required. Thus, to evaluate the distribution patterns of fish in Korea, sampling was conducted on a national scale at 720 sites over a 6-week period in 2009. A total of 124 fish species in 27 families were identified; Zacco platypus and Zacco koreanus of the Cyprinidae were the dominant and subdominant species, respectively. Of the species found, 46 (37.1%) were endemic and 4 (3.2%) exotic; of the latter, Micropterus salmoides and Lepomis macrochirus were widely distributed. Upon canonical correspondence analysis (CCA), both altitude and biological oxygen demand (BOD) were highly correlated with CCA axes 1 and 2, respectively. This explained 62.5% of the species–environment relationship. Altitude and stream order were longitudinally related to species distribution. The numbers of both total and endemic species gradually increased as streams grew in size to the fourth–fifth-order, and decreased in sixth-order, streams. Overall, fish communities were stable throughout the entire watershed, whereas some species showed site-specific occurrence patterns due to the paleogeomorphological characteristics of Korean peninsula. However, various anthropogenic activities may negatively affect fish communities. Therefore, both short- and long-term sustainable management strategies are required to conserve native fish fauna.