Aims: The incidence of severe gastrointestinal toxicity (GIT) following fluorouracil treatment is common and can result in poor clinical outcomes. Currently there is no diagnostic marker to predict a patient’s GIT risk prior to treatment. Given the key role of proinflammatory cytokine production, including interleukin-1 beta (IL-1) via toll-like receptor (TLR) signalling pathways, in the development of GIT, this pilot study investigated the association between genetic variability in immune signalling on the severity of GIT in patients treated with fluorouracil. Methods: Patients that had completed treatment with 5-fluorouracil or capecitabine at Flinders Medical Centre, South Australia, were identified through pharmacy records and invited to participate in the study. Treatment-induced GIT data was collected from case note review. Genomic DNA was isolated from saliva of pre-treated patients and analysed for variants in the following genes using a customised Sequenom MassArray assay: IL-1B, IL-6, IL-10, TNF-a, TGF-b, TLR2, TLR4, MD2, MYD88, BDNF, CRP, ICE. Linear regression with stepwise reduction created a model describing combined genetic variants that were associated with GIT, and receiver operator curves (ROC) determined the performance of the model in predicting GIT. Results: Twenty-seven patients participated in the study, of which 8 had severe GIT (Grade ≥ 3 NCI CTCAE or necessitating a dose reduction). The model that included genetic variability in TLR2 and CRP (individual variant P = 0.022 and 0.326, respectively) was best related to severe GIT, such that the ROC area under the curve from this model was 0.84. Conclusions: This pilot study has revealed for the first time that immune genetic variability of TLR2 and CRP is predictive of patient risk of severe GIT. This study is ongoing in a larger patient population, and expanding to include other immune genetic loci.