Various fluorescence tracers were assessed for their applicability for simultaneously measuring fuel distributions of different volatility classes. Tracers were chosen to show significantly different boiling behaviour representing three volatility classes of non-fluorescing multi-component fuels. Fluorescence properties of the markers were investigated using a heated static high-pressure cell with respect to emission behaviour, temperature and pressure dependence and quenching influences. A combination of ketonic and aromatic tracers appeared to be ideal for simultaneous imaging purposes since fluorescence is emitted in separate spectral regions with little overlap. Simultaneous measurements of the fuel distribution of two volatility classes were performed in a port fuel injected engine showing significant differences in the fuel distributions of low and mid boiling fractions in early stages of compression. The results obtained from fluorescence imaging are compared to fuel spatial distributions gained via tomographic line-of-sight measurements using near infrared absorption which allows the application to commercial fuels without requiring additional marker components.