Objectives Increased SCEs frequencies in human lymphocytes are an indicator of spontaneous chromosome instability and could be influenced by different exogenous and endogenous factors. In this study, we evaluated the influence of age, sex, smoking habits, and genetic polymorphisms on the background levels of SCEs in peripheral blood lymphocytes. Methods Two hundred‐thirty healthy Italian subjects were recruited. Data about age, sex and smoking habits were recorded. Subjects were also genotyped for GSTT1, GSTM1, GSTP1 A/G, CYP1A1 Ile/Val, CYP2C19 G/A, ERCC2/XPD Lys751Gln, XRCC1 Arg194ATrp, XRCC1 Arg399Gln, and XRCC1Arg208His gene polymorphisms. Results The frequency of SCEs/cell was 5.15 ± 1.87, with females showing a significantly higher SCEs value with respect to males (5.36 ± 2.10 and 4.82 ± 1.39, respectively). Smokers showed significantly increased levels of SCEs with respect to nonsmokers (5.93 ± 1.75 and 4.70 ± 1.79, respectively) whereas no differences were observed between heavy and light smokers. Age correlated with the RI value (P = .01) but not with the SCEs frequency (P = 07), although the 31‐40 age group showed a significantly lower SCEs frequency with respect to the other age groups. A significant association was also found between GSTP2C19‐AA, GSTT1‐null, GSTM1‐null, ERCC2/XPD Gln751Gln, and XRCC1 His208His genotypes, and higher frequencies of SCEs. Conclusion We describe the association between some phase I, phase II, and DNA‐repair gene polymorphisms with increased SCEs frequencies, reinforcing the importance of genetic analysis in biomonitoring studies. Sex and age were found to be important endogenous factors that affect the level of genomic damage and the replicative capacity of cells, respectively.