Repetitive DNA sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of such sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and molecular characterization of a novel repetitive DNA sequence from the genome of Citrus limon. Digestion of C. limon DNA with MboI produced a prominent fragment of approximately 600 bp. Southern blotting revealed a ladder composed of DNA fragments that are multimers of the 600 bp Mbo I band. This suggests that MboI isolated a novel satellite, named C. limon satellite DNA 600 (CL600). Methylation analyses using Sau3AI-MboI isoschizomers demonstrated that most cytosine residues in the GATC sites of this element were methylated in C. limon. This sequence was also found in related citrus plants, like grapefruit and orange, and the hardiest close relatives of Citrus, such as kumquat and trifoliate orange.