A tracer-based single-line PLIF imaging technique using a unique optical configuration that allows simultaneously viewing the bulk-gas and the boundary layer region has been applied to an investigation of the naturally occurring thermal stratification in a HCCI engine. Thermal stratification is critical for HCCI engines, because it determines the maximum pressure rise rate which is a limiting factor for high-load operation. The investigation is based on the analysis of temperature maps that were derived from PLIF images, using the temperature sensitivity of fluorescence from toluene introduced as tracer in the fuel. Measurements were made in a single-cylinder optically accessible HCCI engine operating under motored conditions with a vertical laser-sheet orientation that allows observation of the development of thermal stratification from the cold boundary layers into the central region of the charge. Sequences of temperature maps acquired at various crank angles were used to analyze the temporal evolution of thermal stratification.The temperature maps showed that the thermal stratification in the bulk-gas was not due to a consistent gas flow coming from the cylinder walls or to the transport of isolated cold gas pockets. Rather it is a consequence of cold turbulent structures extending from the firedeck and the piston-top. Furthermore, the cold structures extending from the firedeck appear to be especially important, since they are bigger and penetrate further toward the central region of the charge than those from the piston top. Comparison with previous measurements made in the horizontal mid-plane with the same optical engine showed a good agreement. However, these side-view measurements extend this previous work by highlighting new patterns which were impossible to identify with a bottom-view setup. Beyond the qualitative analysis of temperature fields, quantitative measurements of the thermal stratification are also provided both in terms of magnitude and characteristic dimensions of cold pockets.