Background Current research on the physical fitness test scores of high school students is more localized within provinces and cities, with few multicenter, large-sample-size, domain-wide research results. The prevalence of blended online and offline teaching in schools of all types has inevitably prolonged the already long sedentary time of high school students. Whether this trend has some impact on the physical health of high school students is an urgent need for a region-wide study. Objective To investigate and analyze the levels of physical fitness test scores and related influencing factors of urban and rural high school first- and second-year students in seven regions of China, and to explore the empowering paths for the balanced improvement of physical fitness levels of first- and second-year high school students. Methods Stratified randomized cluster sampling method was used to conduct stratified sampling according to urban counties and townships and villages in high school schools in seven regions (East China, South China, North China, Central China, Northwest China, Southwest China, Northeast China) of 31 provinces (municipalities directly under the central government and autonomous regions) of China from January to March 2019, and then randomized clusters of 1 high school school in urban counties and 1 high school school in townships and villages in total of 62 schools were selected respectively. A total of 42 523 freshmen and sophomores in high schools were randomly selected to conduct the research, and the data on students' physical fitness test scores were collected at the same time (BMI reflects the level of physical development; lung capacity reflects the level of physical function; sit-and-reach reflects the level of flexibility; pull-up, 50 m run, standing long jump reflects the level of explosive strength of upper and lower limbs; 1 min sit-up reflects the level of core strength of the torso; 800 m run, 1 000 m run reflects the level of cardiorespiratory endurance), and non-parametric tests and multiple linear regression analysis were used to explore the influencing factors of the level of physical fitness test scores. Results The age distribution of 42 523 high school students was 16.0 (16.0, 17.0) years; 20 074 (47.2%) boys and 22 449 (52.8%) girls, 21 725 (51.1%) in cities, counties and districts, and 20 798 (48.9%) in towns and villages. Comparison of the gender and urban-rural distribution of high school students in the seven regions showed statistically significant differences (PNortheast China>Central China), body mass (Northeast China>North China>Central China), BMI (Central China>Northeast China>North China), lung capacity (Northeast China>Central China>South China), 50 m run (South China>Central China>East China), standing long jump (South China>North China>Northwest China), sit-and-reach (South China>Southwest China>Central China), pull-up (South China>Southwest China>Northwest China), and 1 000 m run (South China>Central China>Southwest China). High school boys in the city, county and districts had higher lung capacity scores than those in the town and country, and lower scores than those in the town and country in the 50 m run, standing long jump, sit-and-reach, pull-up, and 1 000 m run (P0.05). The differences in the scores of high school girls in each physical fitness test in the seven regions are statistically significant (PNortheast China>Northwest China), body mass (Northeast China>North China>Northwest China), BMI (Northeast China>North China>Northwest China), lung capacity (Northeast China>South China>Central China), 50 m run (South China>Central China>East China), standing long jump (South China>North China>Central China), sit-and-reach (Northeast China>Central China>South China), 1 min sit-up (North China>Central China>East China=Southwest China), 800 m run (Northeast>Central China>South China). High school girls in the city and county districts had higher height, body mass, and BMI scores than those in the town and country, and lower scores than those in the town and country in lung capacity, 50 m run, standing long jump, sit-and-reach, 1 min sit-up, and 800 m run (P