This study investigated the residual levels of eight parent neonicotinoid insecticides (p-NEOs) and three NEO metabolites (m-NEOs) in surface waters of river−estuary−ocean systems on a large geographical scale in South China. Imidacloprid, thiamethoxam, acetamiprid, and clothianidin were dominant NEOs in surface waters. High levels of ∑NEOs were found in the upper reaches of Guangzhou section of the Pearl River (PR_GZ), indicating multiple input sources of NEOs in PR_GZ. Additionally, the decreased influence of terrestrial input and human activities was observed along with the flow direction, where the order of ∑NEOs concentration was PR_GZ (inland river, 64.5 ng/L) > Pearl River Estuary (estuary, 33.9 ng/L) > Xisha Islands of the South China Sea (SS_XS, open sea area, 27.0 ng/L); and the level in Northwestern South China Sea (coastal sea, 49.9 ng/L) was also higher than that in open sea area. The estimated annual fluxes of NEOs released into the South China Sea totaled 34.8 ± 20.4 tons, most of which come from the Pearl River basin. To the best of our knowledge, this is the first study to assess the concentrations, distribution, and source analysis of NEOs in river−estuary−ocean systems in South China.