Objective:This study aimed to validate a nutrition knowledge questionnaire appropriate for use in Australia.Design:Nutrition knowledge is essential in establishing and maintaining strategies that reduce the burden of disease and promote wellbeing. The General Nutrition Knowledge Questionnaire (GNKQ) was developed in the United Kingdom in 1999 and validated for Australia in 2008. Changes in national nutrition recommendations and food availability prompted the redevelopment and revalidation of the UK questionnaire in 2016. However, the Australian questionnaire had not been subsequently updated.Setting:Australia.Participants:Content validity was determined using a sample of academic dietitians in Australia (n 8). Face validity was undertaken with retail employees (n 11) whose highest level of education was secondary school. Ninety-three undergraduate nutrition and engineering students at Queensland University of Technology completed the questionnaire for construct validity, and nineteen students were contacted a week later for test-retest reliability.Results:In the 117-scored questionnaire, nutrition students scored consistently higher in each of the four sections and overall (87 %, M 102, IQR 95, 107) compared with engineering students (77 %, M 82, IQR 76, 87·25, P < 0·01). Internal reliability of the questionnaire was high (α = 0·92) as was test-retest reliability (rs = 0·96, ICC2,1 = 0·99). AUS-R NKQ determined significant differences between individuals with known higher levels of nutrition knowledge and obtained high validity, reliability and consistency within an Australian sample.Conclusions:AUS-R NKQ refined through this research is valid and would be an appropriate questionnaire for assessing the effectiveness of nutrition knowledge-based interventions for public health programmes, clinicians and researchers.