The aim of this study was to determine gender differences in the level of motor skills and effects of a multi-year exercise program on the level of motor skills in 161 preschool children (5-6 yo). Patricipants were deployed into one control and three experimental groups. Motor skills were assessed with the Bruininks-Oseretsky Test of Motor Proficiency (BOT-2). To determine difference in scores for each BOT-2 tests between control and experimental groups, one-way ANOVA was used for girls and boys separately while two-way ANOVA was employed to determine difference between the gender in the overall BOT-2 score. The results indicates that one-year multilateral exercise program has a positive effect on the level of motor skills in preschool children. Interestingly, additional years of participation in exercise program yielded in maintenance of acquired motor skills level. Also, exercise program affected more girls than preschool boys considering both individual and composite BOT-2 scores. According to the findings of this study, presented exercise program could have potential benefits on multilateral development of the motor skills in preschool children which could facilitate balance of locomotor and manipulative skills. Therefore, integration of multilateral program intended for preschool children could be considered for implementation within the kindergarten curriculum.