New and modified recombinant factor IX (rFIX) products are in development and accurate potency estimation is important to ensure the consistency of production and efficacy of these therapeutics. Collaborative study data obtained during the replacement of the 3rd International Standard (IS) for FIX concentrate suggested that there was a discrepancy between potency estimates for rFIX using clotting and chromogenic methods, when the rFIX candidate was measured against the plasma-derived FIX (pdFIX) IS. This study explores potential chromogenic and one-stage clotting method discrepancies in more detail. Five batches each of rFIX and pdFIX were assayed against the 4th IS FIX concentrate (a pdFIX) by activated partial thromboplastin time (APTT) one-stage clotting assay and specific functional chromogenic assay. The potency of rFIX by chromogenic assay was consistently around 70% of the one-stage clotting potency (average 78 and 108 IU mL(-1) respectively). These differences were not observed with pdFIX, which had similar potencies (average 96 IU mL(-1) ) by each assay method. In addition, different APTT reagents yielded different potency estimates for rFIX when assayed against the pdFIX IS, with a variation of up to 23%. In all cases, the differences were largely resolved when a rFIX reference was used as the standard. This study highlights some of the challenges associated with assay of rFIX products in the laboratory and that careful consideration needs to be given to the choice of reference material used. This is especially important with the imminent arrival of new and modified rFIX products.