Polymorphisms of the upstream stimulatory factor 1 (USF1) have been associated with carotid artery intima-media thickness and coronary atherosclerotic lesions. Unstable carotid plaque is an atherosclerotic change of vascular morphology that has been correlated with cerebrovascular ischemic symptoms. Associations of three single nucleotide polymorphisms of the USF1 gene with total unstable carotid plaque area (CPA) were investigated in Chinese atherosclerotic stroke patients. We recruited 668 atherosclerotic stroke patients and 602 controls. Total unstable CPA values were measured by ultrasound. Genotypes were analyzed using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) or mismatched PCR-RFLP. A significant difference in total unstable CPA was found for rs2516838 and rs2516839 genotypes (P = 0.039 and 0.046, respectively) in atherosclerotic stroke patients with unstable carotid plaque. Furthermore, in multiple logistic regression analysis adjusted by age, sex, BMI, hypertension, smoking status, glucose, total cholesterol, triglycerides, high-density lipoprotein-cholesterols, low-density lipoprotein-cholesterols and high-sensitivity C-reactive protein, significant associations were seen between the total unstable CPA values and genotypes of the rs2516838 or the rs2516839 in these patients. The rare allele C of rs2516838 or rare allele A of rs2516839 could predict relative low total unstable CPA values. The rs2516838 and rs2516839 polymorphisms of USF1 influence total unstable CPA in atherosclerotic stroke patients, which might be new markers to predict the risk of recurrence for this disease.