A major challenge in the treatment of cancer is dealing with intrinsic and/or acquired chemoresistance and with the development of metastatic lesions. The underlying mechanisms of chemoresistance are complex as a consequence of cancer heterogeneity, such as different tumour tissue origin, inter-tumour heterogeneity between patients and intra-tumour heterogeneity within cell populations of tumours. Classifying tumours according to gene expression-based molecular subtypes predicts the response to therapy and has been proposed to innovate towards personalized therapy. We here highlight the molecular subtypes observed in gastrointestinal cancers. In addition, we discuss the implication of long non-coding RNAs (lncRNAs), a class of RNAs without protein-coding sequence and over 200 nucleotides long, in chemoresistance of gastrointestinal cancers. Furthermore, with the development of single-cell RNA sequencing technologies 10 years ago, it has become clear that intercellular transcriptome heterogeneity of similar cell types may contribute to intra-tumour heterogeneity. Single-cell transcriptome profiling may identify specific cell phenotypes prone to develop chemoresistance that previously remained undistinguished by global gene expression or cell morphology. CRISPR/Cas9 methods may permit to elucidate the role of lncRNAs in chemoresistance at a single-cell level. The single-cell approach may take cancer treatment a step closer towards personalized, or even cell-specific, therapy.