Abstract Both UV spectrophotometry and dynamic light scattering techniques present substantial limitations in characterizing mixed colloid size distributions, challenging their transport studies. We developed a synthetic DNA‐labeling/qPCR approach to distinguish mixed colloids (0.05, 0.5, and 5 μm) transport in porous media. Contrary to single‐size systems where larger colloids (5 μm) were more retained, mixed‐size systems showed higher retention of smaller colloids (0.05 μm). Scanning electron microscopy and filtration analyses revealed size‐dependent heteroaggregation, where smaller colloids deposited on larger ones, forming pore‐blocking aggregates. HYDRUS‐1D simulations identified three governing factors: pore size, surface potential, and media heterogeneity. These findings demonstrate: (a) mixed colloid transport cannot be predicted from monodisperse system behavior and (b) high‐resolution tracking is essential for accurate mixed colloid transport prediction. This work provides critical insights for assessing colloid‐facilitated contaminant transport in groundwater systems.

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