The Sogamoso River basin, a major tributary of the Magdalena River in Colombia, is influenced by agricultural, industrial, and energy-related activities that have promoted the accumulation of trace metals, posing a significant environmental risk. While the presence of these metals in soils and sediments has been documented, their dynamics in water and suspended particulate matter (SPM) are critical to understanding their transport and ecological impact. This study evaluated concentrations of Cd, Pb, and Hg in SPM and their relationship with partitioning mechanisms associated with organic carbon and nitrogen content. Water samples collected at 10 sites along the basin were filtered through 0.45 μm membranes. Cadmium and lead were determined by graphite furnace atomic absorption, while total mercury was measured following EPA Method 7473 using a RA-915LAB Direct Mercury Analyzer. Organic carbon and nitrogen data were incorporated to examine associations with trace metals in SPM. Concentrations ranged from 4.48–63.31 ng/mg SPM for Cd, 31.77–271.66 ng/mg SPM for Pb, and 0.06–0.74 ng/mg SPM for Hg. Results showed a common trend: initial low concentrations in the upper catchment, a rise in areas dominated by cattle ranching and fruit crops, followed by continuous decline. Positive correlations with organic carbon highlight that SPM with higher organic content serves as the main carrier of these contaminants. Findings suggest that trace metals in the Sogamoso River are predominantly bound to fine suspended fractions, favoring downstream transport to the Magdalena River and enhancing ecotoxicological risk.