Background: Vincristine is a key component of many childhood cancer treatment regimens. Pharmacodynamic parameters such as clinical efficacy and toxicity may be influenced by polymorphisms ofCYP3A.Aim: The aim of this study was to documentCYP3A5genotype, vincristine pharmacokinetics (PK) and neurotoxicity profile for 50 children with cancer and determine whether, in a population of Australian children, theCYP3A5genotype influenced the pharmacodynamics of vincristine as reflected by peripheral neurotoxicity.Methods: Blood for PK analysis was collected after any single dose of vincristine and assayed using high performance liquid chromatography with tandem mass spectrometry detection.CYP3A5*3 and CYP3A5*6genotype was determined using gel‐electrophoresis or automated microfluidic electrophoresis. Neurotoxicity was determined by physical examination.Results: The median age of children sampled was 6.5 years (range 1–16.25). Half the patients received concurrent corticosteroids for acute lymphoblastic leukaemia. Six patients (12%) had experienced grade 3 or 4 neurotoxicity. The median clearance, area under the curve and Cmax of vincristine was 482 mL/min/m2(range 132–698), 49.7 mcg/L.h (16.5–143.1) and 3.5 mcg/L (1.0–31.2), respectively. In contrast to prediction, all but three children were homozygous for wild‐typeCYP3A5*3.NoCYP3A5*6polymorphisms were identified.Conclusions: No correlation was identified between vincristine clearance, vincristine neurotoxicity, age, sex or concomitant steroid therapy. The limited sampling methodology proved acceptable to patients and families and would be suitable for larger scale studies including a wider range of genotypic variants and more detailed prospective evaluation of neurotoxicity.