Zellweger syndrome spectrum (ZSS) comprises a clinically and genetically heterogeneous disease entity, which is caused by mutations in any of the 12 different human PEX genes leading to impaired biogenesis of the peroxisome. Patients potentially suffering from ZSS are diagnosed biochemically by measuring elevated levels of very long chain fatty acids, pristanic acid and phytanic acid in plasma and serum and reduced levels of ether phospholipids in erythrocytes. Published reports on diagnostic procedures for ZSS patients are restricted either to biochemical markers or to defined mutations in a subset of PEX genes. Clarification of the primary genetic defect in an affected patient is crucial for genetic counselling, carrier testing or prenatal diagnosis. In this study, we present a rational diagnostic strategy for patients suspected of ZSS. By combining cell biology and molecular genetic methods in an appropriate sequence, we were able to detect the underlying mutation in various PEX genes within adequate time and cost. We applied this method on 90 patients who presented at our institute, Department of Pediatrics and Pediatric Neurology at Georg August University, and detected 174 mutant alleles within six different PEX genes, including two novel deletions and three new missense mutations in PEX6. Furthermore, this strategy will extend our knowledge on genotype-phenotype correlation in various PEX genes. It will contribute to a better understanding of ZSS pathogenesis, allowing the investigation of the effects of diverse mutations on the interaction between PEX proteins and peroxisomal function in vivo.