Gender differences in cross-sectional relationships between fundamental movement skill (FMS) subdomains (locomotor skills, object-control skills) and physical activity were examined in preschool children. Forty-six 3- to 5-year-olds (25 boys) had their FMS video assessed (Test of Gross Motor Development II) and their physical activity objectively monitored (Actigraph 7164 accelerometers). Among boys, object-control skills were associated with physical activity and explained 16.9% (p = .024) and 13.7% (p = .049) of the variance in percent of time in moderate-to-vigorous physical activity (MVPA) and total physical activity, respectively, after controlling for age, SES and z-BMI. Locomotor skills were inversely associated with physical activity among girls, and explained 19.2% (p = .023) of the variance in percent of time in MVPA after controlling for confounders. Gender and FMS subdomain may influence the relationship between FMS and physical activity in preschool children.