Chinese chestnut (Castanea mollissima Bl.) is one of the earliest domesticated and cultivated fruit trees, and it is widely distributed in China. Because of the high quality of its nuts and its high resistance to abiotic and biotic stresses, Chinese chestnut could be used to improve edible chestnut varieties worldwide. However, the unclear domestication history and highly complex genetic background of Chinese chestnut have prevented the efficiency of breeding efforts. To explore the genetic diversity and structure of Chinese chestnut populations and generate new insights that could aid chestnut breeding, heterozygosity statistics, molecular variance analysis, ADMIXTURE analysis, principal component analysis, and phylogenetic analysis were conducted to analyze single nucleotide polymorphism data from 185 Chinese chestnut landraces from five geographical regions in China via genotyping by sequencing. Results showed that the genetic diversity level of the five populations from different regions was relatively high, with an observed heterozygosity of 0.2796-0.3427. The genetic diversity level of the population in the mid-western regions was the highest, while the population north of the Yellow River was the lowest. Molecular variance analysis showed that the variation among different populations was only 2.07%, while the intra-group variation reached 97.93%. The Chinese chestnut samples could be divided into two groups: a northern and southern population, separated by the Yellow River; however, some samples from the southern population were genetically closer to samples from the northern population. We speculate that this might be related to the migration of humans during the Han dynasty due to the frequent wars that took place during this period, which might have led to the introduction of chestnut to southern regions. Some samples from Shandong Province and Beijing City were outliers that did not cluster with their respective groups, and this might be caused by the special geographical, political, and economic significance of these two regions. The findings of our study showed the complex genetic relationships among Chinese chestnut landraces and the high genetic diversity of these resources.