Specific genetic conditions are known to be associated with high risk of venous thromboembolism. This genetic basis varies widely between ethnic groups. We investigated the distribution of four inherited polymorphisms in 113 unselected Tunisian blood donors by using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. The allele frequencies of Factor V Leiden (FVL), prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 677C>T, and MTHFR 1298A>C mutations were 3, 0.9, 30, and 31%, respectively. The MTHFR 677C>T polymorphism was influenced by age. Twenty‐nine of the 113 blood donors demonstrated more than one genetic markers. Hyperhomocysteinemia was found in 12 subjects, and it was statistically associated to the MTHFR 677TT genotype. Principal component analysis allowed disclosing the resemblance between Mediterranean populations. Our findings may be helpful for population genetics study, and provide epidemiologic database for further studies in thrombosis field among Tunisians. J. Clin. Lab. Anal. 26:167‐173, 2012. © 2012 Wiley Periodicals, Inc.