Studies on the nature of restriction fragment length polymorphisms (RFLPs) were undertaken to characterize the Citrus genome. This type of analysis has not been carried out with any other perennial crop. Citrus reticulata Blanco cv Clementine, C. xparadisi Macf. cv Duncan, and an F1 hybrid (LB 1-21) were used to determine what probe/enzyme combinations revealed polymorphisms in Southern analysis, and a backcross family (LB 1-21x'Clementine') of 65 randomly selected hybrid seedlings was used for some analyses. A majority (73%) of the clones examined from a PstI genomic library appeared to detect single-copy sequences based on RFLP banding patterns, while clones from a cDNA library revealed a lower percentage of single copy sequences. When hybridization stringencies were lowered, 21% of the genomic clones examined revealed greater copy numbers. PstI digestion of 'Duncan' DNA indicated abundant methylation, so the relatively high frequency of multiple-copy sequences observed at moderate stringency cannot be attributed to a lack of methylation of the Citrus DNA. The polymorphisms in banding patterns observed primarily resulted from insertions and/or deletions rather than from base substitutions, and a model is presented to account for the varying patterns obtained from individual probes with different restriction enzymes. Finally, a model for transposon activity in Citrus is proposed, based on observations made during the course of these studies.