Fluorescence in situ hybridization (FISH) is an increasingly powerful tool with a variety of applications in both basic and applied research. With excellent genetic, cytogenetic and molecular maps available, the tomato genome provides a good model to benefit from the full potential of FISH. Tomato chromosomes at mitotic metaphase are small and not particularly suitable for high-resolution FISH. In contrast, chromosomes at meiotic pachytene are about 15 times longer, and easier to identify by their differences in chromosome arm lengths and chromomere pattern. We have developed a technique for preparing chromosomal spreads of young pollen mother cells at mid-prophase I which is suitable for FISH. In a first series of experiments, the hybridization patterns of three classes of repetitive DNA sequences were studied in single and multicolour FISH.