The aim of the this study was to establish age- and gender-specific physical fitness normative values and to compare percentiles and Z scores values in a large, nationwide sample of Greek children aged 6-18 years. From March 2014 to May 2014, a total of 424,328 boys and girls aged 6-18 years who attended school in Greece were enrolled. The studied sample was representative, in terms of age-sex distribution and geographical region. Physical fitness tests (i.e. 20 m shuttle run test (SRT), standing long jump, sit and reach, sit-ups, and 10 × 5 m SRT) were performed and used to calculate normative values, using the percentiles of the empirical distributions and the lambda, mu, and sigma statistical method. Normative values were presented as tabulated percentiles for five health-related fitness tests based on a large data set comprising 424,328 test performances. Boys typically scored higher than girls on cardiovascular endurance, muscular strength, muscular endurance, and speed/agility, but lower on flexibility (all p values <0.001). Older boys and girls had better performances than younger ones (p < 0.001). Physical fitness tests' performances tended to peak at around the age of 15 years in both sexes. The presented population-based data are the most up-to-date sex- and age-values for the health-related fitness of children and adolescents in Greece and can be used as standard values for fitness screening and surveillance systems and for comparisons among the same health-related fitness scores of children from other countries similar to Greece. Schools need to make efforts to improve the fitness level of the schoolchildren through the physical education curriculum to prevent cardiovascular risk.