Background: In type 1 von Willebrand Disease (VWD) patients, von Willebrand Factor (VWF) levels and bleeding symptoms are highly variable. Recently, the association between genetic variations in STXBP5 and STX2 with VWF levels has been discovered in the general population. We assessed the relationship between genetic variations in STXBP5 and STX2, VWF levels, and bleeding phenotype in type 1 VWD patients. Methods: In 158 patients diagnosed with type 1 VWD according to the current ISTH guidelines, we genotyped three tagging-SNPs in STXBP5 and STX2 and analyzed their relationship with VWF:Ag levels and the severity of the bleeding phenotype, as assessed by the Tosetto bleeding score. Results: In STX2, rs7978987 was significantly associated with VWF:Ag levels (beta-coefficient (beta) = -0.04 IU/mL per allele, [95%CI -0.07;-0.001], p = 0.04) and VWF:CB activity (beta = -0.12 IU/mL per allele, [95%CI -0.17;-0.06], p<0.0001). For rs1039084 in STXBP5 a similar trend with VWF:Ag levels was observed: (beta = -0.03 IU/mL per allele [95% CI -0.06;0.003], p = 0.07). In women, homozygous carriers of the minor alleles of both SNPs in STXBP5 had a significantly higher bleeding score than Conclusions: Genetic variation in STX2 is associated with VWF: Ag levels in patients diagnosed with type 1 VWD. In addition, genetic variation in STXBP5 is associated with bleeding phenotype in female VWD patients. Our findings may partly explain the variable VWF levels and bleeding phenotype in type 1 VWD patients.