Introduction. CDKN2A is a polymorphic tumor suppressor gene associated with variants 500 C>G, 540 C>T and 442 G>A (Ala148Thr). The 442 G>A has been associated to melanoma. In this study we analyzed CDKN2A polymorphic variants 500 C>G, 540 C>T and 442 G>A in order to determine whether they are associated with head and neck squamous cell carcinoma (HNSCC) development. In addition we investigated, in a subgroup of patients, the role of polymorphic variants regarding the methylation status of the CDKN2A gene. Materials and Methods. HNSCC Puerto Rican patients (152) and healthy controls (124) were included in this study. Genotyping of the CDKN2A was performed using the Restriction Fragment Length Polymorphisms (PCR-RFLP) Assay. Determination of DNA methylation status was performed using Methylight Assay or Methylation Specific PCR (MSP). Results. Our results show that frequencies of CDKN2A polymorphisms 442 G>A, 500 C>G, and 540 C>T in HNSCC patients differ significantly from the control group (p=0.009, p<0.0001, p=0.02, respectively). Aberrant DNA methylation of CDKN2A promoter region was found in 27 (46%) cases while in 32 (54%) cases the gene promoter was unmethylated. However, there were no significant differences in genotype frequencies between patients with methylated or unmethylated CDKN2A promoter. Conclusions. Differences in frequency of 442 G>A, 500 C>G, 540 C>T CDKN2A polymorphic variants between HNSCC patients and controls might play a role in HNSCC development in the Puerto Rican population. We found no evidence that these polymorphisms modify the methylation status of the CDKN2A promoter in HNSCC patients.Programa de Honor. Recinto de Río Piedras. Universidad de Puerto Rico.