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In an optically accessible single-cylinder engine fueled with hydrogen, acetone planar laser-induced fluorescence (PLIF) and particle image velocimetry (PIV) are used to evaluate in-cylinder mixture formation. The experiments include measurements for engine operation with hydrogen injection in-cylinder either prior to or after intake valve closure (IVC). Pre-IVC injection is used to produce a near-homogeneous mixture for PLIF calibration experiments and to establish a baseline comparison for post-IVC injection. Calibration experiments and a temperature correction allow conversion of the acetone fluorescence signal to equivalence ratio. For post-IVC injection with start of injection (SOI) coincident with IVC, PLIF results are similar to pre-IVC injection. With retard of SOI from IVC, mixture inhomogeneities increase monotonically, with high hydrogen concentration spatially located near the injector and within a smaller volume.