Heavy metal contamination in water bodies is a pervasive and persistent environmental challenge in many parts of the world, especially in developing countries. This study investigates the use of multivariate analysis methods for monitoring variations in water quality along a spatial gradient and for the interpretation of pollution levels at different sampling sites. We assessed the water quality of the Seybouse River and identified possible sources of pollution using three complementary multivariate analysis techniques (PCA, NMDS, and K-means clustering). The results indicate a longitudinal gradient in water quality associated with industrial and agricultural activities in the middle and lower Seybouse River. Physico-chemical and heavy metal analyses show high water turbidity with elevated concentrations of iron and chromium. We show that the contamination stems from four different sources, which can be categorized into different pollution levels. Our results suggest that complementary multivariate methods are a robust approach to identifying and categorizing significant sources of pollution in rivers, enabling the development of future successful water quality management strategies based on water pollution levels. This study highlights the importance of monitoring water quality and taking effective measures to control and mitigate pollution from various sources to ensure the safety of the environment and human health.