Land use plays a significant role in determining the spatial patterns of water quality in the Johor River Basin (JRB), Malaysia. In the recent years, there have been several occurrences of pollution in these rivers, which has generated concerns over the long-term sustainability of the water resources in the JRB. Specifically, this water resource is a shared commodity between two states, namely, Johor state of Malaysia and Singapore, a neighbouring country adjacent to Malaysia. Prior to this study, few research on the influence of land use configuration on water quality have been conducted in Johor. In addition, it is also unclear how water quality varies under different seasonality in the presence of point sources. In this study, we investigated the influence of land use and point sources from wastewater treatment plants (WWTPs) on the water quality in the JRB. Two statistical techniques - Multivariate Linear Regression (MLR) and Redundancy Analysis (RA) were undertaken to analyse the relationships between river water quality and land use configuration, as well as point sources from WWTPs under different seasonality. Water samples were collected from 49 sites within the JRB from March to December in 2019. Results showed that influence from WWTPs on water quality was greater during the dry season and less significant during the wet season. In particular, point source was highly positively correlated with ammoniacal‑nitrogen (NH3-N). On the other hand, land use influence was greater than point source influence during the wet season. Residential and urban land use were important predictors for nutrients and organic matter (chemical oxygen demand); and forest land use were important sinks for heavy metals but a significant source of manganese.