Deep vein thrombosis is a life-threatening development of blood clots in deep veins. Immobility and blood flow stagnancy are typical risk factors indicating that fluid dynamics play an important role in the initiation of venous clots. However, the roles of physical parameters of the valves and flow conditions in deep vein thrombosis initiation have not been fully understood. Here, we describe a microfluidics in vitro method that enabled us to explore the role of valve elasticity using in situ fabrication and characterisation. In our experimental model the stiffness of each valve leaflet can be controlled independently, and various flow conditions were tested. The resulting complex flow patterns were detected using ghost particle velocimetry and linked to localised thrombus formation using whole blood and an aqueous suspension of polystyrene particles. In particular, valves with leaflets of similar stiffness had clot formation on the valve tips whereas valves with leaflets of different stiffness had clot formation in the valve pocket.