The clonal origin of malignancy and hematopoiesis is a principal tenet of modern biology and medicine. This paper describes a highly specific and sensitive assay for the detection of clonality in cells and cell lineages suitable for studies in a large proportion of females. The specific ligase chain and/or ligase detection reactions (LCR/LDR) are utilized at a polymorphic glucose-6-phosphate dehydrogenase (G-6-PD) locus for discrimination of the mRNA transcripts of the active X chromosome. This combination approach circumvents problems encountered with other currently used assays of clonality based either on peptide G-6-PD polymorphism or on DNA methylation differences between the active and inactive X chromosomes. The veracity of this assay was verified by analysis of 19 random healthy females as well as by the study of hemopoietic and nonhemopoietic tissues from a patient with clonal hemopoiesis/polycythemia vera. Furthermore, we demonstrate that the G-6-PD locus used in our clonal assay does not display marked differences in methylation between the active and the inactive X chromosomes.