Introduction: Although considerable progress has been made in breast cancer research over the past few decades, more than 11,000 women died from this disease in 2014 in the UK. De novo or acquired resistance to standard anti-estrogen therapy is one of the main reasons for the high cancer mortality incidence in estrogen receptor positive (ER+) breast tumours. There is evidence that Cancer Stem Cells (CSCs) are the culprits for the lack of response to anti-estrogen treatments. Therefore, this study sought to determine the effects of anti-estrogens on different CSC populations. In addition, we aimed to characterise anti-estrogen resistant CSCs and to investigate their cellular diversity, in order to identify biomarkers that can be therapeutically targeted. Methodology: The proportion of CSCs following anti-estrogen treatment in ER+ cell lines and patient-derived samples (PDS) was assessed using flow cytometry to measure cell surface antibody binding, autofluorescent cells and aldehyde dehydrogenase (ALDH) activity. The stem cell activity of ALDH positive (ALDH pos) cells was assayed using in vivo transplantation and in vitro mammosphere formation assays and their gene expression profile was studied using Affymetrix Arrays. Finally, ALDH pos cellular diversity was investigated at the single cell level using the C1 system and Biomark HD technologies (Fluidigm). Results: ALDH pos cells are enriched in ER+ cell lines and PDS following Tam and Fulv treatment. Although the exact mechanism remains elusive, the ALDH1A3 isoform appears to be important at driving this enrichment in MCF-7 cells. ALDH pos cells from MCF-7 and PDS have a distinct gene expression pattern when compared to ALDH negative cells. Additionally, single cell analysis in the ALDH pos population revealed that they are not a homogeneous cellular compartment but exist in 3 clusters defined by gene expression. One of these clusters is only present after anti-estrogen treatment. Conclusion: Anti-estrogen therapies enrich for cells with CSC properties (ALDH pos cells) which comprise at least 3 distinct populations of cells. Future studies should assess the functional relevance of these cellular subgroups and how to therapeutically target them to improve treatment of anti-estrogen resistant breast cancer.