Impurity partitioning at grain boundaries (GBs) during polycrystalline colloidal crystallization has been investigated via direct observation. Polycrystalline grains have a partitioning behavior similar to that of single colloidal crystals, which follows the Burton, Prim, and Slichter (BPS) model. We have found that impurities segregate at GBs during colloidal crystal growth, and the impurity concentration at GBs (C GB) for various misorientation angles (θ) between adjacent grains and growth rates (V) has been investigated. C GB was found to increase with either increasing θ or V, and also when the size of the impurity is close to that of the host colloid particle. In situ observations reveal that impurities incorporated into GBs are supplied mostly from the impurities segregated at the solid–liquid interface, and C GB and the growth rate show a BPS-like relationship.