The aim of this study was to investigate the effect of different shoe-heel heights on the surface electromyographic (EMG) activity of vastus medialis (VM) and vastus lateralis (VL) during treadmill walking in female patients with patellofemoral pain (PFP). Nineteen women with PFP participated in this research. EMG signals were recorded from the VM and VL of both sides and were compared during the treadmill walking. The subjects walked on a treadmill wearing shoes of three different heel heights: 1 cm, 3 cm and 7 cm. Each subject walked on a treadmill for five minutes at a speed of 2 km/hour with three minutes resting intervals between consecutive trials. The data were analyzed by one-way repeated-measures analysis of variance. The results of the present study indicate that EMG data of the VM and VL of female patients with PFP did improve with an increase in the height of the shoe heel, which were statistically significant. Additionally, the EMG activity of VM increased more dramatically than that of VL associated with the task of walking with high-heeled shoes on the treadmill. This study suggests that the type of high-heeled shoes is related to the VM and VL muscle activation patterns contributing to knee joint pathologies in female patients with PFP.