Background Most validations of paediatric glomerular filtration rate (GFR) estimating equations using standardized creatinine (CR) and cystatin C (CYS) assays have comprised relatively small cohorts, which makes accuracy across subgroups of GFR, age, body mass index (BMI) and gender uncertain. To overcome this, a large cohort of children referred for GFR determination has been established from several European medical centres. Methods Three thousand four hundred eight measurements of GFR (mGFR) using plasma clearance of exogenous substances were performed in 2218 children aged 2–17 years. Validated equations included Schwartz-2009 CR /2012 CR/CYS/CR+CYS , FAS CR/CYS/CR+CYS , LMR CR , Schwartz-Lyon CR , Berg CYS , CAPA CYS , CKD-EPI CYS , Andersen CR+CYS and arithmetic means of the best single-marker equations in explorative analysis. Five metrics were used to compare the performance of the GFR equations: bias, precision and three accuracy measures including the percentage of GFR estimates (eGFR) within ± 10% (P 10 ) and ± 30% (P 30 ) of mGFR. Results Three of the cystatin C equations, Berg CYS , CAPA CYS and CKD-EPI CYS , exhibited low bias and generally satisfactory accuracy across all levels of mGFR; CKD-EPI CYS had more stable performance across gender than the two other equations. Among creatinine equations, Schwartz-Lyon CR had the best performance but was inaccurate at mGFR < 30 mL/min/1.73 m 2 and in underweight patients. Arithmetic means of the best creatinine and cystatin C equations above improved bias compared to the existing composite creatinine+cystatin C equations. Conclusions The present study strongly suggests that cystatin C should be the primary biomarker of choice when estimating GFR in children with decreased GFR. Arithmetic means of well-performing single-marker equations improve accuracy further at most mGFR levels and have practical advantages compared to composite equations.