All cancers emerge following the selection and subsequent expansion of malignant clones. Underpinning this clonal outgrowth is a reservoir of genetic and non-genetic intra-tumoral heterogeneity (ITH) that supports the functional diversification of cancer cells within an individual tumor and contributes to therapeutic resistance. Whilst the evolutionary principles imparted by genetic ITH are becoming increasingly clear, little is known about the non-genetic mechanisms that contribute to ITH and malignant clonal fitness. To identify factors that contribute to non-genetic ITH and drive tumor progression and clonal outgrowth, we developed Single-cell Profiling and LINeage TRacing (SPLINTR), a synthetic lentiviral barcoding strategy that couples lineage identity to transcriptomic information at single cell resolution. Using a clinically relevant mouse model of acute myeloid leukaemia (AML) we find that malignant clonal dominance is an intrinsic and heritable property that is facilitated by the repression of antigen processing and presentation pathway genes, mature granulocyte markers and the increased expression of Secretory Leukocyte Peptidase Inhibitor (Slpi), which we genetically validate as a novel regulator of AML. Compared to extramedullary sites, leukaemia initiating capacity is most enriched in malignant cells resident within the bone marrow microenvironment. Finally, we demonstrate that the non-genetic transcriptional program that defines leukaemia initiating clones can be inherited by clonal progeny and is conserved within a subset of cells comprising dominant clones across serial transplantations. Together these data provide fundamental insights into the transcriptional processes that underpin malignant clonal fitness that may inform future therapeutic strategies aiming to limit the impact of non-genetic ITH on tumor development.