A substantial number of genes mutated in human cancers encode components of the cell cycle processes. As the G1/S transition in the cell cycle is a finely regulated biological process, we hypothesized that sequence variations in the promoter region of the related genes might indeed lead to abnormal expression, thus predisposing the individuals carrying these genetic variants to cancer. In this report, we screened the promoter regions of 16 cell cycle checkpoint genes for DNA variants and assessed the functional impact of these promoter region single nucleotide polymorphisms (pSNPs) by combining in silico analysis and in vitro functional assays. We identified 127 pSNPs including 90 with predicted impact on putative binding sites of known transcription factors. Eleven pSNPs were selected for electrophoresis mobility shift assays because of their association with predicted gains of binding sites, and nine pSNPs showed differential allelic shifts in at least one cell line tested. Following the subcloning of the promoter regions into a gene reporter system, we found that at least four promoter haplotypes associated with CCND1, E2F1, HDAC1 and RB1 significantly influenced transcriptional activity in an allele-specific manner. Although the biological significance of these observations still remains to be demonstrated, the expected variability of expression levels in key cell cycle components might influence individual's risk of cancer.