The Skudai River has experienced a general decline in water quality over the last several years due to agricultural practices, economic development, and other human activities in the river catchment. The spatial trend of water quality index (WQI) and its sub-indexes are important for determining the locations of major pollutant sources that contribute to water quality depletion in the Skudai and its tributaries. In this study, we have developed WQI for eight sections of the Skudai watershed. Ammonia-nitrogen (NH₃-N) was identified as a major pollutant downstream from the Skudai, with the lowest WQI index (i.e. 38). WQI for the Skudai (natural) was 94, i.e., Class I (very clean) category of river water quality. The Senai River has WQI value of 85 and Class II category (slightly polluted). However, the Kempas River, which was in suburban parts of the Skudai watershed, had WQI of 53 (Class III, polluted). The Melana and Danga rivers were also polluted rivers with WQI of 69 and 57, respectively, in Class III (polluted). Overall water quality in the Skudai and its tributaries was downstream of the river. The study also assessed water quality of the Skudai and its tributaries from other water quality parameters such as conductivity, turbidity, temperature, total dissolved solids, total phosphorous, and nitrogen, which were not part of the WQI formula developed by the Department of the Environment (DOE), Malaysia. The study found that Department of Education (DOE) formula for WQI was not effective in water quality assessment as many important parameters such as nutrients, heavy metals, and fecal coliform (or E. Coli) were missing in the WQI formula.