Colorectal cancer (CRC) is the second leading cause of cancer death in Australia. One of the key challenges with therapeutic strategies is defining inter-individual differences that impact a patients’ response to therapy. A personalised cancer therapy approach would transform the management of CRC. As organoids recapitulate many of the in vivo characteristics of the original tumour tissue, we aim to develop preclinical models for predicting patient-specific responses to treatment using patient-derived organoids (PDOs). In this study, we examined the histological, molecular and phenotypic characteristics including the stem cell signature in 11 PDOs and patient-matched primary tumours. The PDOs recapitulate the histopathological characteristics of their primary tumours displaying distinct morphological differences between patients. Immunohistochemistry and gene expression analyses for markers of different categories of stem cell populations including the LGR5+, reserve and revival stem cells revealed similar expression patterns between PDOs and primary tumours. In response to chemotherapeutic treatment with 5-Fluorouracil (5-FU), PDOs displayed a 3-fold difference in sensitivity, and interestingly more resistant organoid lines were associated with higher expression of stem cell markers. This study provides insight into the inter-tumoural heterogeneity in stem cell signatures and the clinical relevance of PDOs as preclinical models to predict drug sensitivity.