Physical and genetic mapping of the phytoplasma chromosome can be a useful tool in a genome sequencing project in order to assemble the in silico-predicted contigs robustly. Mapping consists of four distinct steps: preparation of phytoplasma chromosomes from infected plants, single- and double-digestion of chromosomes with rare-cutting restriction enzymes, separation of large DNA fragments by pulsed-field gel electrophoresis, and hybridization with various genetic markers. Materials and methods needed for each step are described and the technique is illustrated using the flavescence dorée phytoplasma genome map as an example.