BackgroundLaryngeal squamous cell carcinoma (LSCC) represents one of the principal tumors of the head and neck. Human papillomavirus (HPV) and Epstein–Barr virus (EBV) are considered risk factors for the development and the clinical prognosis of LSCC. High levels of p16INK4aare suggested as a surrogate marker of HPV or EBV infection in some head and neck tumors but in LSCC is still controversial. Furthermore, pRb expression may be considered an additional biomarker but it has not been clearly defined. This work aimed to compare the expression of pRb and p16INK4aas possible biomarkers in tumor tissues with and without infection by EBV or different genotypes of HPV from patients with LSCC.MethodsTumor samples from 103 patients with LSCC were previously investigated for the presence and genotypes of HPV using the INNO-LiPA line probe assay and for the infection of EBV by qPCR. p16INK4aand pRb expression was assessed by immunohistochemistry.ResultsOf the 103 tumor samples, expression of p16INK4awas positive in 55 (53.4%) and of this, 32 (56.1%) were positive for HPV whereas 11 (39.3%) were EBV positive but both without a significantly difference (p> 0.05). pRb expression was positive in 78 (75.7%) and a higher frequency of this expression was observed in HPV negative samples (87.0%) (p= 0.021) and in high-risk HPV negative samples (85.2%) (p= 0.010). No difference was observed when comparing pRb expression and EBV infection status (p> 0.05).ConclusionOur results support the suggestion that p16INK4ais not a reliable surrogate marker for identifying HPV or EBV infection in LSCC. On the other hand, most of our samples had pRb expression, which was more frequent in tumors without HPV, suggesting that pRb could indicate HPV negativity. However, more studies with a larger number of cases are required, including controls without LSCC and evaluating other molecular markers to determine the real role of p16INK4aand pRb in LSCC.