The polymerase chain reaction (PCR) clonality assay based on the principle of random X chromosome methylation in females provides a potentially important tool in both cancer research and diagnostics. This assay, however, has not been compared to the standard Southern blot assay and is limited by the rate of heterozygosity of the X-linked phosphoglycerate kinase (PGK) and androgen receptor genes, the only two genes yet described with which this technique may be used. Using 46 marrow and blood specimens from females with and without hematological malignancies, the PCR and Southern blot methods of clonality were compared. In addition, a new technique based on the highly polymorphic fragile X (FMR1) locus was examined. The rate of heterozygosity was 25% for the PGK gene and 45% for the FMR1 gene. In the PCR assay, 7 of 8 and 11 of 14 normal control specimens showed a polyclonal methylation pattern in the PGK and FMR1 genes, respectively. Of the malignant specimens, 17 of 17 and 17 of 18 showed a monoclonal methylation pattern in the PGK and FMR1 genes, respectively. The Southern blot and PCR assay gave similar results with regards to the PGK gene. It is concluded that the PCR and Southern blot clonality assays are comparable with regards to the PGK gene and that both the PGK and FMR1 genes may be reliably used in the determination of clonality. The methods, however, are limited by the skewed methylation patterns seen in hematological specimens in a significant number of normal females.