Uterine fibroids are conventionally defined as clonally derived benign tumours from the proliferation of a single smooth muscle cell. We have previously identified fibroblast-like cells in fibroids, the presence of which raises the question as to whether all cells within the fibroid have the same clonal origin. The first aim of this study was to develop a fluorescence-activated cell sorting-based method to isolate different cell types from human myometrium and fibroid tissues. Secondly, we aimed to use X-chromosome inactivation analysis to determine the clonality of cell sub-populations isolated from myometrial and fibroid tissues. Human myometrium and fibroid tissues were collected from women undergoing hysterectomy. Immunohistochemistry and flow cytometry confirmed that in addition to smooth muscle cells, fibroblasts constitute a significant proportion of cells in human myometrium and fibroid tissues. Fluorescence-activated cell sorting based on CD90 and ALDH1 reliably separated cells into 3 myometrial and 4 fibroid sub-populations; smooth muscle cells, vascular smooth muscle cells and 2 fibroblast subsets. Clonality was first determined by X-chromosome inactivation using the classic DNA methylation sensitive HUMARA assay. Data from this assay were highly variable, with only a quarter of samples meeting the definition of clonal fibroid and non-clonal myometrium. However, using an RNA-based X-chromosome inactivation HUMARA assay, we were able to demonstrate clonality of all cellular constituents of most fibroids. Our results confirm that most fibroids are derived from a single cell, and for the first time demonstrate that these clonal cells differentiate into fibroblast and smooth muscle cell sub-population as the fibroid grows.