Disinfection by-products (DBPs) in drinking water pose a threat to human health. This study, for the first time, investigated the distribution of 22 DBPs in the drinking water of 32 cities along the Yangtze River Basin and Yellow River Basin and assessed their health effects on the Chinese population. The concentrations of total DBPs in drinking water from the Yangtze River Basin (11.0−122.6 μg/L) are essentially consistent with those in the Yellow River Basin (12.0−122.2 μg/L). The levels of trihalomethanes (THMs) and haloacetic acids (HAAs) in most cities were within limits set by the "Standards for Drinking Water Quality" (GB 5749−2022). Additionally, haloacetaldehydes (HALs) and haloacetonitriles (HANs) were found to be the primary driving factors for cytotoxicity and genotoxicity. The Yangtze River Basin and Yellow River Basin exhibited medium to high carcinogenic risks from DBPs for different population groups. However, no potential non-carcinogenic risks from DBPs to human health (HQs < 1) have been observed. In conclusion, controlling DBPs in drinking water, especially unregulated DBPs, is necessary to reduce their harm to human health.