The analysis of high molecular weight DNa by pulsed field gel electrophoresis (PFGE) is an essential step for physical genome mapping, Bacterial Artificial Chromosome (BAC) or Yeast Artificial Chromosome (YAC) library. construction and gene isolation. One of the main draw-backs to the success of physical mapping techniques is the need to obtain good quality high molecular weight DNA.We have developed a rapid, reliable method for the preparation of high molecular weight genomic DNA from sweet orange (Citrus sinensis (L.) Osb.) by adapting a nuclear isolation protocol. The nuclei are then embedded directly in agarose plugs and the DNA is cleaved by rare cutting restriction enzymes after pre-treatment. The DNA fragments are separated by PFGE. DNA fragments greater than 2 Mb are obtained.Within-gel hybridization was used instead of classical Southern blotting with sweet orange cDNA probes. The same gel can be re-used several times after probes are removed. These results were compared to those obtained with a classical method of DNA preparation (ground leaf powder).