The Kali River is a significant source of surface water as well as the main tributary of River Hindon that flows through major cities of western Uttar Pradesh, India. It flows throughout the urban and industrial regions; hence, it carries various amounts of pollutant. Therefore, a study was conducted to examine spatial-temporal variations in river water quality by determining physicochemical variables and heavy metal concentrations at seventeen sampling stations (S-1-S-17) throughout the river stretch. Various physicochemical variables, namely pH, EC, TDS, turbidity, BOD, COD, TH, TA, Ca, Mg, Na, K, HCO3-, Cl-, SO42-, NO3-, and PO43- were higher in summer than in winter. The order of mean metal concentrations was Fe > Pb > Mn > Ni > Zn > Cu > Cr > Cd. The relationships among measured physicochemical variables and pollution index were examined. Furthermore, multivariate statistical methods were used to assess spatial-temporal variation in water quality to identify current pollution sources and validate results. Water quality index and comprehensive pollution index indicated that the Kali River was less polluted from S-1 to S-8. However, downstream sampling sites were polluted. Pollution starts from S-9 and drastically increases at and beyond S-13 because of effluents from industries and sugar mills in Muzaffarnagar. The study suggests cleaning the downstream region of river to restore human health and flora and fauna in the river ecosystem.