The ribosomal RNA (rRNA)-encoding genes (rDNA) in flax, estimated to be present in about 2400 copies per diploid nucleus, have been reported as a single homogeneous repeat unit of 8.6 kb. In situ hybridization analysis indicated that these genes were located at a single site on one pair of chromosomes. However, an analysis of a flax variety, CI 1303, has revealed heterogeneity in the intergenic spacer of the rDNA repeat unit. A genetic analysis of rDNA inheritance in two flax lines, Stormont Cirrus and CI 1303, has again supported the observation that there is a single rDNA locus in this plant species. Screening of four different genomic libraries made in methylation-sensitive and -insensitive systems, and the analysis of 40 phage clones, demonstrate a much higher number than that expected of junctions between rDNA and non-rDNA. Direct evidence of rRNA-encoding genes being present in tandem comes from a few phage clones that contain more than two rDNA repeats. The evidence presented here indicates that rDNA, although present at a single locus in tandem arrays, may be interrupted frequently by other non-rDNA sequences, thus giving rise to questions about their organization into long tandem arrays.