Hyperoxaluria, one of the major risk factors for calcium oxalate urolithiasis and nephrocalcinosis, causes significant morbidity and mortality and should therefore be detected and treated as soon as possible. An early, consequent and adequate evaluation, but also a distinction between primary (PH) and secondary hyperoxaluria (SH) is therefore essential. We evaluated the usefulness of three consecutive 24-h urine collections under different diets [usual diet, (A), low oxalate diet, (B), high oxalate diet, (C)] to prove SH, or to find evidence of PH by changes in urinary oxalate excretion (Uox). We retrospectively analyzed results from 96 pediatric patients (47 females and 49 males, age 3–18 years) who presented with a history of nephrolithiasis, nephrocalcinosis and/or persistent hematuria in whom hyperoxaluria was found in an initial urine sample. The typical pattern of SH was found in 34 patients (mean Uox (A) 0.85 ± 0.29, (B) 0.54 ± 0.15 and (C) 0.95 ± 0.28 mmol/1.73m2/d). PH was suspected in 13 patients [(A) 1.21 ± 0.75; (B) 1.47 ± 0.51 and (C) 1.60 ± 0.82 mmol/1.73m2/d], but genetically proven only in 1/5 patients examined. No hyperoxaluria was found in 16 patients. Data were inconclusive in 33 patients. Urine collection under different diets is helpful to diagnose secondary hyperoxaluria and may provide evidence, that urinary oxalate excretion is normal. We have now established this procedure as our first diagnostic step before further, more extensive and more expensive evaluations are performed.