This chapter describes chromosome microdissection, with emphasis placed on the dissection of plant chromosomes using a laser dissection system. It also describes a chromosome preparation method for microdissection and structure of the laser system. The chapter shows how to dissect and clone the chromosome segment using several plant chromosomes. It explains a probe-labeling procedure for in situ hybridization steps using laser dissected plant materials. In situ hybridization determines whether direct cloned DNA originates from the chromosome region. Direct cloning and simultaneous direct labeling reduce the effort of cloning and labeling the specific DNA sequences. Nucleus and chromosome samples prepared by ordinary cytological methods provide a good template for DNA amplification by a polymerase chain reaction (PCR) method. For in situ hybridization, the chromosome samples are prepared by the Enzymatic Maceration/Air-Drying Method (EMA) method without staining and air-dried at room temperature for 3 to 7 d before use.