Polyploidy is an important evolutionary process in plants, but much remains to be learned about the evolution of gene expression in polyploids. Evolution and expression of the 18S-5.8S-26S ribosomal gene family was investigated at homeologous loci in the Glycine subgenus Glycine perennial soybean polyploid complex, which consists of several diploid genomes that have formed allopolyploids in various combinations, often recurrently. A semiquantitative PCR method targeting the internal transcribed spacer (ITS) of the 18S-5.8S-26S nuclear ribosomal DNA (nrDNA) was used to survey the ratio between homeologous repeats in polyploid genomes and to test for preferential expression of homeologous nrDNA loci. Most natural polyploids possess one predominant nrDNA homeolog in their genome. Analysis of F2 segregation in an artificial cross suggested that in some plants, most or all repeats at one homeologous locus have been lost, whereas in other plants two loci remain, but both have been homogenized by concerted evolution. In most natural allopolyploids harboring a relatively balanced ratio of homeologs, one homeolog was expressed preferentially, but in the majority of plants, low levels of transcription could be detected from the other homeolog. Individuals within some tetraploid taxa varied as to which homeolog was expressed preferentially. In some plants, the degree of preferential expression also varied among tissues. Preferential expression was absent in synthetic polyploids and in some artificial diploid hybrids, suggesting that nucleolar dominance is not necessarily a direct result of hybridization or polyploidization. The establishment of preferential expression in Glycine allopolyploids appears to be either stochastic within lineages or genotype specific.