Abstract Background and Aims Cystatin C is recommended for use along with creatinine in estimating glomerular filtration rate (eGFR) when precise estimates are needed for clinical decision-making. Although eGFR based on both creatinine and cystatin (eGFRcr-cys) is the most accurate in research studies, it is uncertain as to whether this remains true in real-world settings, particularly when there are large discordances between eGFR based on creatinine (eGFRcr) and that based on cystatin C (eGFRcys). Method We included 6185 adults from the Stockholm Creatinine Measurements (SCREAM) project referred for plasma clearance of iohexol in Stockholm, Sweden, who had 9404 concurrent measurements of creatinine, cystatin C and iohexol clearance. The performance of eGFRcr, eGFRcys and eGFRcr-cys was assessed against mGFR with bias, P30 and correct classification of GFR strata. We stratified analyses within three categories: eGFRcys at least 20% lower than eGFRcr (eGFRcys < eGFRcr), eGFRcys within 20% of eGFRcr (eGFRcys ≈ eGFRcr) and eGFRcys at least 20% higher than eGFRcr (eGFRcys > eGFRcr). Bias was expressed as the median difference in estimated GFR minus mGFR, with negative biases indicating underestimation of mGFR. P30 described the percentage of individuals with eGFR within 30% of mGFR. Correct classification of GFR categories was defined as agreement of eGFR and mGFR categories using the KDIGO GFR categories (<15, 15-29, 30-44, 45-59, 60-89 and ≥90 ml/min/1.73 m2). Results eGFRcr and eGFRcys were similar in 4226 (45%) of samples, and there all three estimating equations displayed similar performance (Table 1). In contrast, eGFRcr-cys was much more accurate in cases of discordance. For example, when eGFRcys < eGFRcr (47% of samples), median biases were 15.0 (overestimation), −8.5 (underestimation) and 0.8 ml/min/1.73 m2 for eGFRcr, eGFRcys and eGFRcr-cys, respectively; P30 was 50%/73%/84%, respectively; and correct classification was 38%/45%/62%, respectively. When eGFRcys > eGFRcr (8% of samples), median biases were −4.5, 8.4, and 1.4 ml/min/1.73 m2. Findings were consistent among individuals with cardiovascular disease, heart failure, diabetes mellitus, liver disease and cancer (Figure 1). Conclusion When large discordances between eGFRcr and eGFRcys are found in clinical practice, eGFRcr-cys is more accurate than either eGFRcr or eGFRcys.