The purpose of this study was to compare the effect of shoulder abduction angles, dominant and non-dominant arm and external loading on co-contraction ratio during arm abduction in scapular plane in overhead athletes and non-athletes. 10 swimmers, 10 handball players and 10 tennis players and ten non-athletes voluntarily participated in this study. Electromyography activity of shoulder muscles (middle and anterior deltoid; upper, middle and lower trapezius; infraspinatus, serratus anterior and latissimus dorsi) during dynamic and static arm abduction in 3 different angles (0-45º in 1second and holding it for 3 seconds, 0-90º in 2 seconds and holding it for 3 seconds, 0-135º in 1 second and holding it for 3 seconds) was recorded for dominant and non-dominant arm in loading and non-loading conditions. Co-contraction ratio of shoulder muscles was calculated for both groups. Findings indicated external loading, shoulder dominance and abduction angles had significant effect on shoulder muscles co-contraction ratio during (0-45º, 0-90º, 0-135º) and holding shoulder elevation in scapular plane (45º, 90º, 135º),  also there was significant difference in shoulder muscles co-contraction ratio among four groups during and holding elevation in scapular plane (p≤0.05). Significant differences in shoulder muscles co-contraction ratio among athletes group and between athletes and non-athletes may be related to sport demands and adaptation to exercises and extensive use of upper limb as well.