Hypertrophic Cardiomyopathy (HCM), a common and clinically heterogeneous disease characterized by unexplained ventricular myocardial hypertrophy and a high risk of sudden cardiac death, is mostly caused by mutations in sarcomeric genes but modifiers genes may also modulate the phenotypic expression of HCM mutations. The aim of the current study was to report the frequency of single and multiple gene mutations in a large French cohort of HCM patients and to evaluate the influence of polymorphisms previously suggested to be potential disease modifiers in this myocardial pathology. We report the molecular screening of 192 unrelated HCM patients using denaturing high-performance liquid chromatography/sequencing analysis of the MYBPC3, MYH7, TNNT2 and TNNI3 genes. Genotyping of 6 gene polymorphisms previously reported as putative HCM modifiers (5 RAAS polymorphisms and TNF-α -308 G/A) was also performed. Seventy-five mutations were identified in 92 index patients (48%); 32 were novel. MYBPC3 mutations (25%) represent the most prevalent cause of inherited HCM whereas MYH7 mutations (12%) rank second in the pathogenesis. The onset age was older in patients carrying MYBPC3 mutations than in those with MYH7 mutations. The MYBPC3 IVS20-2A>G splice mutation was identified in 7% of our HCM population. Multiple gene mutations were identified in 9 probands (5%), highlighting the importance of screening other HCM-causing genes even after a first mutation has been identified, particularly in young patients with a severe phenotype. No single or cumulative genetic modifier effect could be evidenced in this HCM cohort.