Diagnosis of factor XIII (FXIII) deficiency (FXIIID) as a rare bleeding disorder is a challenge worldwide. Thus, in the present study, we used different methods including two molecular methods for detection of FXIIID. This study was conducted on individuals suspected to FXIIID. All individuals were checked by two routinely used methods of clot solubility test in Iran and two other clot solubility tests as well as FXIII activity and antigen assays. Molecular analysis was performed by PCR-restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system (T-ARMS)-PCR for only FXIIID mutation in southeast Iran (p.Trp187Arg), previously associated with severe FXIIID. Out of 151 individuals, 26 had abnormal clot solubility test with all four methods. PCR-RFLP revealed that 27 patients were homozygotes for p.Trp187Arg, whereas 12 were heterozygotes. Molecular analysis revealed that in routinely used clot solubility combinations, two homozygotes (∼8%) were missed, whereas in two other combinations, one patient (∼4%) was missed. One false positive result was observed in routinely used methods, whereas further combinations don’t have false positive. T-ARMS-PCR had three discrepancies with PCR-RFLP and sequencing confirmed that the results of T-ARMS-PCR were false. FXIII antigen assay diagnosed all homozygotes, whereas in FXIII activity assay, two homozygotes had higher than 5% FXIII activity that inconsistent with severe deficiency. It seems that clot solubility test is not enough sensitive and specific and molecular analysis is the most reliable method for detection of FXIIID in areas such Iran with one or few specific mutations.