Referral bias can influence the results of studies performed at tertiary-care centres. In this study, we evaluated demographic and microbiological factors that influenced referral of patients with Gram-negative bloodstream infection (BSI). We identified 2919 and 846 unique patients with Gram-negative BSI in a referral cohort of patients treated at Mayo Clinic Hospitals and a population-based cohort of Olmsted County, Minnesota, residents between 1 January 1998 and 31 December 2007, respectively. Multivariable logistic regression analysis was used to determine factors associated with referral. Elderly patients aged ⩾80 years with Gram-negative BSI were less likely to be referred than younger patients [odds ratio (OR) 0·43, 95% confidence intervals (CI) 0·30–0·62] as were females (OR 0·63, 95% CI 0·53–0·74). After adjusting for age and gender, bloodstream isolates of Escherichia coli (OR 0·50, 95% CI 0·43–0·58) and Proteus mirabilis (OR 0·49, 95% CI 0·30–0·82) were underrepresented in the referral cohort; and Pseudomonas aeruginosa (OR 2·26, 95% CI 1·70–3·06), Enterobacter cloacae (OR 2·31, 95% CI 1·53–3·66), Serratia marcescens (OR 2·34, 95% CI 1·33–4·52) and Stenotrophomonas maltophilia (OR 17·94, 95% CI 3·98–314·43) were overrepresented in the referral cohort. We demonstrated that demographic and microbiological characteristics of patients with Gram-negative BSI had an influence on referral patterns. These factors should be considered when interpreting results of investigations performed at tertiary-care centres.