In this study we performed the CHEK2 c.1100delC mutation analysis in 1046 breast cancer patients and 730 unaffected control individuals. The mutated allele was found in 3 out of 688 unselected sporadic breast cancer patients and in 1 out of 358 familial/early onset breast cancer patients. Two mutations were identified in a cohort of 730 controls. Our results support the finding that frequency of CHEK2 c.1100delC mutation varies among different populations. Based on our results, genotyping of CHEK2 c.1100delC mutation in clinical settings in the Czech Republic could not be recommended.