Freckles, one of the common defects in blades used in heavy-duty gas turbines, hugely deteriorate the mechanical properties and liability of blades under service conditions. The thermal-solutal convection theory is a widely adopted formation mechanism, but few solid experimental pieces of evidence have been reported. Here, the grain microstructure in freckle chains taken from four different Nickel-based superalloys with either single-crystal or directionally solidified alloy is analyzed for the first time. The relationship between the internal stress and the misorientation throughout the freckle chains is studied by means of state-of-the-art electron microscopy. The results supply new experimental evidence of the thermal-solutal convection theory, which is further supported by the fact that borides at the boundary are randomly orientated to alloys. Therefore, this research enriches the methodology of freckle study, providing new insight into the formation mechanism of casting defects.