Information about the regional distribution of health-related physical fitness status is necessary in order to tailor public health interventions, and due to a number of behavioral health risks caused by the increasing sedentary lifestyle. This study aimed to find differences between Croatian children's health-related physical fitness profiles in urban and rural areas. The sample for this study consisted of 2431 fifth-grade students (1248 boys and 1183 girls) from urban and rural areas of Croatia. The mean age of participants was 11.3 +/- 6.1 years. The differences between the health-related physical fitness of school children from urban and rural areas was computed using series of univariant analysis of variance and canonical discriminant analysis. The reliability of the tests was determined by Cronbach's alpha coefficients. Urban boys and girls significantly differ in body height from rural boys and girls. Body mass index and body fat percentage are slightly higher in the urban boys and girls but they do not differ significantly. Urban children perform significantly better in the 20 m dash, standing long jump and timed sit-ups. Urban and rural boys and girls do not differ significantly in the flexibility. This study determined if selected levels of urbanization affected the physical fitness status of children in Croatia. The results suggest that the differences in children's health-related physical fitness profiles are due to the level of urbanization.