Microplastic pollution in the marine environment is a global issue. Recently, in South Korea, there has been active research on the establishment of testing methods and investigation of distribution characteristics of microplastics not only in marine environments but also in freshwater systems. In particular, it is necessary to study the distribution of microplastics such as rivers and lakes, which are drinking water sources, to affect the water quality environment, management, and countermeasures. This study identified the distribution of microplastics in the mainstream and tributary of the Han River water system. The freshwater, sediment, and biological species samples were pre-treated accordingly, and microplastics larger than 20 µm were analyzed using μ-FT-IR. As a result of the inter-laboratory study, in the same way as in this study, it was possible to confirm that the trusted recovery rate was 63.6%. The survey results indicated that the number of microplastics per location ranged from 1,173 to 8,940 particles/L in freshwater samples, 3,800 to 192,000 particles/kg in sediment samples, and 34 to 384 particles/individual in fish samples. The most commonly found microplastic materials were PP, PE, and PTFE. The size distribution of plastics appeared in the following order: 45~100 µm, 100~300 µm, 20~45 µm, and greater than 300 µm, with predominantly fragmented shapes observed.