BACKGROUND: Little is known about health characteristics and the physical activity (PA) patterns in children attending preschools. The objective of this study was to describe the gender differences in relation to body mass index (BMI), motor skills (MS) and PA, including PA patterns by the day type and time of day. Additionally, the between-preschool variation in mean PA was estimated using the intraclass correlation. METHODS: We invited 627 children 5-6 years of age attending 43 randomly selected preschools in Odense, Denmark. Aiming and catching MS was assessed using subtests of the Movement Assessment Battery for Children (Second Edition) and motor coordination MS was assessed by the Kiphard-Schilling body coordination test, Körperkoordination Test für Kinder. PA was measured using accelerometry. The PA patterns were analysed using mixed models. RESULTS: No gender differences in the BMI or norm-referenced MS risk classification, or the average weekly PA level or patterns of PA were observed. However, boys performed better in the aiming and catching score (p < 0.01) and in the motor coordination score (p < 0.05) on average. Girls performed better in the balance subtest (p < 0.001). Relative to the norm-referenced classification of MS, the Danish sample distribution was significantly well for aiming and catching but poorer for the motor coordination test.The total sample and the least active children were most active on weekdays, during preschool time and in the late afternoon at the weekend. However, a relatively larger decrease in PA from preschool to weekday leisure time was observed in children in the lowest PA quartile compared to children in the highest PA quartile. Finally, the preschool accounted for 19% of the total variance in PA, with significant gender differences. CONCLUSIONS: Results of this study could provide a valuable reference material for studies monitoring future trends in obesity, MS and PA behaviour in Denmark and other countries.Knowledge about sources of variation in PA among preschool children is scarce and our findings need to be replicated in future studies. A potentially important finding is the large between-preschool variation in PA, indicating that especially girls are very susceptible to the environment offered for PA during preschool attendance.