The aim of the present study was to investigate and benchmark the level of general nutrition knowledge in elite Australian athletes (EA) against a similar aged community (CM) and criterion sample with dietetic training (DT). EA (n 175), CM (n 116) and DT (n 53) completed the General Nutrition Knowledge Questionnaire (GNKQ), which assesses four domains (sections A–D) of general nutrition knowledge (section A: dietary guidelines; section B: sources of nutrients; section C: choosing everyday foods; section D: diet–disease relationships). Age, sex and education level were collected in all groups, and athletic calibre and sport type (team or individual) in EA. Dietitians and nutrition scientists (n 53) re-examined the GNKQ for content validity, resulting in instrument revision (R-GNKQ; ninety-six items). Psychometric assessment (internal consistency: Cronbach-α; test–retest: Spearman rank correlation) was performed in a sub-sample (n 28). Independent t tests, ANOVA and ANCOVA (χ2 for categorical variables) were used to assess between-group differences. DT scored higher than EA and CM in all sub-sections and overall (P < 0·005). EA scored lower than CM in GNKQ for section B (P < 0·005) and overall (P < 0·005), and in R-GNKQ for section B (P < 0·005), section C (P < 0·005), section D (P = 0·006) and overall (P < 0·005). Overall score was influenced by age (P = 0·036 for GNKQ: P = 0·053 for R-GNKQ), sex (P = 0·016 for GNKQ: P = 0·003 for R-GNKQ) and athletic calibre (P = 0·029 for R-GNKQ only), but not level of education, living situation or ethnicity. EA and CM performed best on section A and worst on D. EA had lower overall general knowledge scores than CM. This was significantly influenced by age and sex.