The presence of antibiotics as emerging micropollutants in the aquatic environment has been raising health concerns for a number of years. Macrolides (MLs) are a large class of widely-used antibiotics, but there is a lack of information on their distribution and risks in surface waters across the central and western regions of China. To clearly describe the pollution characteristics and risks of MLs in Weihe River (the largest tributary of the Yellow River), analysis of 5 typical MLs was conducted using a newly developed LC−MS/MS method for 50 water samples collected over three seasons during 2021–2022. The results revealed that the area showed comparable ML concentrations with other regions worldwide. However, the ML concentrations were much lower than those in the river during 2016 from a previous study. Furthermore, concentrations exhibited significant seasonal variation, with highest concentrations in the winter. Along the main stream of the Weihe River, the sampling sites close to the wastewater treatment plants (WWTPs) and livestock farms exhibited higher concentrations of MLs, indicating the significant contribution from WWTPs and animal husbandry to the emission of MLs. The risk quotients (RQs) suggested that the ecological risks associated with MLs were most pronounced in winter among the three seasons, with erythromycin posing a high or medium risk to algae at all sampling sites. The results of this study will be of importance towards the goal of understanding the presence of these emerging contaminants in surface waters and any required risk reduction measures.