In camera-based motion tracking, the target structure has a significant impact on the tracking accuracy. In the present research, a new method is proposed for the optimization of the location of five markers on a visible target for the camera. The proposed objective function for positioning of markers is equal to the total distance of each marker from all of the planes formed from the combination of triads made of other markers. To avoid the symmetry of the target structure, which makes the labeling of markers on the image impossible, a constraint is applied that guarantees a minimum difference in the distance between the pairs of markers. The genetic algorithm is exploited for maximizing the proposed objective function with the mentioned constraint. The experimental results obtained from the positioning of markers on the target using the proposed method (with or without the application of the constraint) and other methods are evaluated. These methods are compared in terms of error in estimating 3D pose and tracking speed in the presence of noise. It is notable that the results confirm the applicability of the proposed method in the positioning of markers.