Creep resistant low-alloyed 2.25Cr-1Mo-0.25V steel is typically applied in hydrogen bearing heavy wall pressure vessels in the chemical and petrochemical industry. For this purpose, the steel is often joined via submerged-arc welding. In order to increase the reactors efficiency via higher operating temperatures and pressures, the industry demands for improved strength and toughness of the steel plates and weldments at elevated temperatures. This study investigates the influence of the post weld heat treatment (PWHT) on the microstructure and mechanical properties of 2.25Cr-1Mo-0.25V multi-layer weld metal aiming to describe the underlying microstructure-property relationships. Apart from tensile, Charpy impact and stress rupture testing, micro-hardness mappings were performed and changes in the dislocation structure as well as alterations of the MX carbonitrides were analysed by means of high resolution methods. A longer PWHT-time was found to decrease the stress rupture time of the weld metal and increase the impact energy at the same time. In addition, a longer duration of PWHT causes a reduction of strength and an increase of the weld metals ductility. Though the overall hardness of the weld metal is decreased with longer duration of PWHT, PWHT-times of more than 12 h lead to an enhanced temper resistance of the heat-affected zones (HAZs) in-between the weld beads of the multi-layer weld metal. This is linked to several influencing factors such as reaustenitization and stress relief in the course of multi-layer welding, a higher fraction of larger carbides and a smaller grain size in the HAZs within the multi-layer weld metal.