The purpose of this study was to compare the change in electromyography (EMG) activity in the gluteus maximus (G-max) and the gluteus medius (G-med) in subjects with and without chronic ankle instability (CAI) during three functional postures. Twenty four females were recruited for this study. Subjects were assigned into two groups: with CAI (n1=12) and without CAI (n2=12). The assessment postures were rotational squat, one leg stand above a gradient and crossed leg-sway. Electromyographic activities of the G-max and the G-med were recorded using surface EMG and was normalized using the maximal voluntary isometric contraction elicited using a manual muscle testing. Independent t-test was used to determine the statistical differences between two groups during the three functional postures. The comparisons of the three posture between two groups were performed using a one-way repeated analysis of variance. A Bonferroni adjustment used for post hoc analysis. The activation of EMG on G-max performing the one leg stand above a gradient and crossed leg-sway in subjects with CAI is significantly higher than normal group (p<.05). The activation of EMG on the G-max during the rotational squat was significantly increased, compared to those of the one leg stand above a gradient and crossed leg-sway (p<.05). The activation of EMG on G-med performing three exercise at CAI is significantly higher than normal group (p<.05). The activation of EMG on the G-med during the crossed leg-sway was significantly increased, compared to the rotational squat (p<.05). This study provides valuable information for clinician who research CAI.