Display omitted •A method was established for simultaneously determining 68 glucocorticoids.•Sixteen natural and synthetic glucocorticoids were first reported in river samples.•Concentrations of natural metabolites were up to 36 times those of cortisol itself.•Halogenated GCs are the main contributors to GC activities in the studied waters.•GCs could be used as potential indicators of sewage input. Glucocorticoids (GCs) have been increasingly reported to have adverse effects on aquatic organisms, but the lack of comprehensive analytical methods for a broad number of GCs has limited the effective management of pollution by these molecules in surface and coastal waters. In this study, we developed an original analytical method for simultaneously monitoring 25 natural GCs, and 43 synthetic GCs (4 hydrocortisone types, 6 acetonide types, 8 betamethasone types, 14 halogenated esters, and 11 labile prodrug esters) in water samples. Of the river samples investigated, 15 natural and 25 synthetic compounds were detected with the concentrations ranging from 0.13 ng/L (11-epitetrahydrocortisol) to 433 ng/L (cortisone) and from 0.05 (clobetasol) to 94 ng/L (prednisolone), respectively. Thirteen natural metabolites of cortisol (CRL) were first detected, and their concentrations were up to 36 times higher than that of CRL. Hydrocortisone-type GCs were the dominant synthetic compounds (≤154 ng/L), followed by halogenated esters (≤81 ng/L), acetonide type GCs (≤57 ng/L), betamethasone type GCs (≤32 ng/L), and labile prodrug esters (≤22 ng/L). Considering the relative potencies for detected GCs compared to dexamethasone, halogenated esters predominantly contributed to the GC activities in the samples. Notably, this is the first report of the halogenated esters 11-oxo fluticasone propionate (OFP) and cloticasone propionate (CTP) in environmental waters. Untreated wastewater is the main source of GCs in the studied waters, and the concentration ratios between natural and synthetic GCs can be used as potential indicators of sewage input. Because of the high detected concentrations and bioactivity potency of halogenated GCs, they are the main contributors to GC activities in the studied waters, and deserved more study in the future.