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Chemical reaction kinetics plays an important role in homogeneous charge compression ignition (HCCI) combustion. In order to control the combustion process, the underlying mechanism of auto-ignition must be explored, especially for the HCCI combustion using negative valve overlap (NVO) strategy, in which the residual gas affects the auto-ignition of next cycle remarkably. In this research, experimental research was carried out in a single cylinder gasoline engine equipped with an in-cylinder sampling system which mainly consists of a special spark plug, a sampling tube and a high-speed electromagnetic valve. In-cylinder charge was sampled at compression stroke and analyzed by FTIR with two types of fuel injection strategy, such as port fuel injection (PFI) solely and port fuel injection combined with injection during negative valve overlap (PFI & NVO-Injection).