Abstract Background Cohort studies are among the most robust of observational studies but have issues with external validity. This study assesses threats to external validity (generalizability) in the European QUALity (EQUAL) study, a cohort study of people >65 years of age with Stage 4/5 chronic kidney disease. Methods Patients meeting the EQUAL inclusion criteria were identified in The Health Improvement Network database and stratified into those attending renal units, a secondary care cohort (SCC) and a not primary care cohort (PCC). Survival, progression to renal replacement therapy (RRT) and hospitalization were compared. Results The analysis included 250, 633 and 2464 patients in EQUAL, PCC and SCC. EQUAL had a higher proportion of men compared with PCC and SCC (60.0% versus 34.8% versus 51.4%). Increasing age ≥85 years {odds ratio [OR] 0.25 [95% confidence interval (CI) 0.15–0.40]} and comorbidity [Charlson Comorbidity Index ≥4, OR 0.69 (95% CI 0.52–0.91)] were associated with non-participation in EQUAL. EQUAL had a higher proportion of patients starting RRT at 1 year compared with SCC (8.1% versus 2.1%; P < 0.001). Patients in the PCC and SCC had increased risk of hospitalization [incidence rate ratio 1.76 (95% CI 1.27–2.47) and 2.13 (95% CI 1.59–2.86)] and mortality at 1 year [hazard ratio 3.48 (95% CI 2.1–5.7) and 1.7 (95% CI 1.1–2.7)] compared with EQUAL. Conclusions This study provides evidence of how participants in a cohort study can differ from the broader population of patients, which is essential when considering external validity and application to local practice.