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BackgroundThe Movement Assessment Battery for Children-second edition (MABC-2) Age Band 1 is widely used to identify preschoolers with motor difficulties. Despite unsatisfactory construct validity of the original three-factor model, MABC-2 (manual dexterity, aiming and catching, and balance), previous research has not considered possible age and gender differences throughout the entire preschool period.AimThe aim of this study was to verify the construct validity of the MABC-2 Age Band 1 in a population of Czech preschoolers with respect to age and gender.MethodsUsing data from 510 Czech preschoolers (3–6 years; 4.9 ± 1.1 years), confirmatory factor analyses (CFA) were used for each age category and gender.ResultsThe goodness-of-fit indices of CFA supported the original three-factor model of the MABC-2 only in 3- and 4-year-old children, and in boys (3–6 years). Low factor loadings and ceiling effects of several test items (Drawing Trail, Walking Heels Raised, and Jumping on Mats) seem to be a probable cause of weak fit indices in 5- and 6-year-old children and in girls (3–6 years).ConclusionThese results suggest that the MABC-2 can be a valid tool for assessing motor development and identifying motor difficulties among 3- to 4-year olds, and generally fits better for preschool boys in the Czech Republic. However, in 5- to 6-year olds, ceiling effects and a low power of discrimination was found for the Drawing Trail, Walking Heels Raised, and Jumping on Mats tests. Therefore, the three-factor model is not appropriate for all preschoolers, and separate norms should be established for each age and gender.