In the context of toluene laser-induced fluorescence (LIF) thermometry, the two common LIF detection strategies, namely one-color and two-color detection, have been simultaneously applied to compare each strategy’s ability to accurately resolve thermal gradients during an engine cycle within an optically accessible internal combustion (IC) engine. Temperature images are obtained from high-speed toluene LIF measurements and are combined with high-speed particle image velocimetry. The combination with flow data and Mie scattering images facilitates the interpretation of differences between the toluene LIF detection strategies. Two-color temperature images are limited in their ability to detect thermal gradients near the end of compression due to larger precision uncertainties. Local regions of cold gases in the two-color images are better identified with the guidance of the one-color images when homogeneous toluene mixtures preside. During expansion, large differences exist between one- and two-color temperature images and likely caused by local mixture fraction heterogeneities that bias the one-color detection strategy. Toluene condensation occurs during the expansion and exhaust stroke and causes local mixture fraction heterogeneities in the combustion chamber. Liquid toluene is in contact with solid surfaces and crevices of the combustion chamber and can evaporate during compression or expansion causing both local temperature and mixture stratification. This work demonstrates the advantage of high-speed imaging and use of multiple image diagnostics to reveal the development of natural temperature and mixture stratification in a motored IC engine. This work also suggests that natural temperature stratification typically regarded from gas-wall heat transfer may also be caused by liquid droplet evaporation on solid surfaces. Such phenomenon, however, is expected to be pertinent for all modern-day engine operating systems.