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In this study, the effects of fuel injection on in-cylinder flow under various injection conditions were investigated using particle image velocimetry measurements in a two-cylinder, direct-injection spark-ignition, optically accessible engine with a spray-guided injection system. Various injection timings and pressures were applied to intensify the turbulence of in-cylinder flow. Simple double-injection strategies were used to determine how multiple injections affect in-cylinder flow. The average flow speed, turbulent kinetic energy, and enhancement level were calculated to quantitatively analyze the effects of fuel injection. Fuel injection can supply additional momentum to a cylinder. However, at an early injection timing such as 300° before top dead center, in-cylinder flow development could be disturbed by fuel injection due to piston impingement and interactions between the spray and air.