Background Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management.Aim To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype.Methods Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories.Results FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories.Conclusion FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.