Introduction: The purpose of this study was to examine the effect of play based on fundamental movement skills on locomotion and manipulative skills acquisition on different genders. Methodology: 25 preschool children (3.5 to 6 year-old) randomly assigned to two groups: intervention group (N=12) and control group (N=13) on different genders (11 boys and 14 girls). For assessing manipulative and locomotion skills, Test of Gross Motor Development-2 (TGMD-2) was employed that include run, gallop, hop, leap, horizontal jump and slide for locomotion skills and striking a stationary ball, stationary dribble, kick, catch, overhand throw and underhand roll for manipulative skills. The intervention group had 30 one-hour sessions (three days a week for 10 weeks) fun play for acquisition fundamental movement skills but control group received no systematic training and just did their ordinary activities. Shapiro-Wilk test was used for checking data normality. To analyze data, ANOVA with repeated measures and SPSS software were employed. Results: The results showed that play based on fundamental movement skills significantly improved both locomotion and manipulative skills. In the locomotion skills, the main effects of gender (P<0.01) and training (p<0.05) were significant and in the manipulation skills also, the main effects of gender (p<0.001) and training (p<0.001) were significant. The results showed superiority of training group and boys than control group and girls. Discussion: Findings indicate this physical play can improve gross motor skills and also showed this kind of play is more effective for manipulative skills than locomotion because manipulative skills need eye-hand and eye-foot coordination and are weaker than other fundamental movement skills and need more accuracy and intervention and girls need more attention.