Fluorochrome sequential labelling of mineralizing tissues is commonly used in different fields of clinical and basic research. Recently we improved polychrome fluorescent sequential labelling of bone by applying spectral image analysis to discriminate seven different fluorochromes. Although basic mineralization processes of bone and teeth follow comparable principles, the respective tissues differ in terms of matrix composition and mineral assembly. The aim of this study therefore was to investigate the feasibility of this new technique for polychrome sequential labelling of teeth and to demonstrate the advantages in the field of dentistry. Furthermore, the exact labelled area of each fluorochrome could be measured, even in regions of overlapping fluorochromes. The technique presented may provide a basis for further investigations of mineralization processes of different anatomical dental structures.