A new method based on real time PCR with FRET and melting curve analysis was developed for the detection of factor XIII A subunit (FXIII-A) Val34Leu polymorphism (V34L). The rapid, simple method is well applicable for large-scale analysis. In whole plasma the onset of FXIII activation is determined by fibrin formation, while the rate of activation is modulated by V34L. FXIII levels were elevated in patients with peripheral artery disease (PAD) and the elevation was more evident in females. The severity of PAD did not show significant correlation with FXIII levels. FXIII levels in the upper tertile conferred a 2-2.3-fold increased risk of PAD to females, i.e., elevated FXIII could be considered a gender specific risk factor of PAD. FXIII-A V34L did not influence the risk of PAD. No association was revealed between the risk of non-fatal atherothrombotic ischemic stroke (AIS) and FXIII-A Val34Leu genotype. In contrast, in females homozygous presentation of Leu34 allele represented a more than 3.0-fold increased risk of AIS with fatal outcome. FXIII-A V34L does not influence the occurrence of AIS, but has a gender specific effect on the severity of its outcome.