Negative valve overlap (NVO) is a valve strategy employed to retain and recompress residual burned gases to assist HCCI combustion, particularly in the difficult regime of low-load operation. NVO allows the retention of large quantities of hot residual burned gases as well as the possibility of fuel addition for combustion control purposes. Reaction of fuel injected during NVO increases charge temperature, but in addition could produce reformed fuel species that may affect main combustion phasing. The strategy holds potential for controlling and extending low-load HCCI combustion.The goal of this work is to demonstrate the feasibility of applying two-wavelength PLIF of 3-pentanone to obtain simultaneous, in-cylinder temperature and composition images during different parts of the HCCI/NVO cycle. Measurements are recorded during the intake and main compression strokes, as well as during the more challenging periods of NVO recompression and re-expansion. To improve measurement quality, effects of diagnostic uncertainty and fluorescence interference are quantified. Temperature, fuel, and EGR images are captured for a range of NVO operating conditions, including main and NVO fuel-injection timings as well total load. The results demonstrate that the diagnostic is capable of providing information useful for the study of HCCI/NVO engine operation.