A multi-stack module consisting of a number of stacks connected in series and parallel serves as a basis for installation of MW-scale vanadium flow battery system in grid storage applications. Due to the existence of stack-to-stack variation in resistance, the module performance can be notably limited by an inappropriate module layout that magnifies the impact of stack resistance variation through series and parallel connections. To understand the layout effect on performance, an in-depth investigation is conducted for an eight-stack 250 kW module in this study. Based on experimental measurements, the correlation of module layout to performance is firstly revealed on both the 250 kW module and a laboratory mini-module. Subsequently, 35 different layouts are specified for the 250 kW module and their performance is fully evaluated by means of development of dynamic models for the module. Simulation results prove that the module charging capacity can be effectively improved by grouping stacks with similar resistances into the same branch and be further promoted by optimizing the flow rate for the stack with the largest resistance. The present study offers not only mechanistic insights into the importance of module layout but a cost-effective way to evaluate the module performance as well.