The growing concern of aquatic heavy metal (HM) pollution is dependent on the toxic nature of its bio-available form. Thus, bio-availability is guided by the HM fractionation in water. This study was therefore conducted to evaluate the spatial impact on physicochemical fractionation of HM (Zn, Mn, Cu, and Fe) in the waters of the Hirakud reservoir in India. Speciation along different pores using a filtration technique was adopted to fractionate the HMs in water samples. The result suggests that the water of the study area is polluted with Cu (0.22-0.35mg/L), Mn (0.15-0.23mg/L), and Fe (1.90-3.10mg/L) that have crossed their permissible limits while Zn (0.17-0.97mg/L) was within the permissible standard. When studied for physical partitioning, the right dyke was comparatively more polluted than the left dyke. While the water samples were dominated by the dissolved fraction of heavy metals, it was construed that a large proportion of the HMs are in bio-available form. Further, a distinct impact of spatial variation on metal fractionation was also evident in the study with PCA revealing site-specific behaviour. Therefore, it can be concluded that multiple anthropogenic activities lead to the distribution and fractionation of HMs in water.