A major QTL of maize stalk rot resistance, molecular markers linked with the major QTL and their uses are disclosed. The QTL provided by the present invention locates in chromosome 10, between the molecular markers SSR334 and SSR58. 59 pairs of molecular markers of the major QTL are also provided. The molecular markers provided by the present invention are significantly correlated with stalk rot resistance in statistical level, and can be used in molecular marker-assisted breeding of maize stalk rot resistance to produce resistant varieties or improve stalk rot resistance of maize varieties.