The influence of Co content on stacking fault energy (SFE) of the γ matrix in four Ni–Co base superalloys, including newly developed alloys, has been studied by utilizing high-resolution transmission electron microscopy. The results indicated the SFE was not linear with Co content of the γ matrix. The lowest SFE could be attained at around 34.0 at.% Co. This effect was attributed to variation of electron holes, saturated Co content in the matrix, and the effect of Co on the partition coefficient of other alloying elements. A high density of twins was related to low SFE and could improve the mechanical properties.