We studied the expression of p16(INK4a) in a series of HPV-negative laryngeal squamous cell carcinomas and assessed its association with prognosis. Forty-five patients with laryngeal carcinoma were included in the study. Clinicopathological features and prognosis were reviewed. p16(INK4a) protein expression was analysed through immunohistochemistry. We analysed messenger RNA (mRNA) in 25 cases through quantitative reverse transcription polymerase chain reaction. HPV status was assessed by PCR using three different protocols based on MY09/11 and GP5/6 primers. Four out of 45 (9 %) cases overexpressed p16(INK4a) protein and showed a tendency to worse survival that was significant for stages I-III (log-rank p value = 0.001). Expression of p16(INK4a) mRNA was high in 12 out of 25 (48 %) cases using an arbitrary cut-off level. All tumours were HPV negative with all three detection methods. A CDKN2A mutation was found in eight cases. One case with a missense and one with a frameshift mutation showed p16(INK4a) protein expression by immunohistochemistry. Six out of seven (86 %) mutated but only 6 out of 18 (33 %) non-mutated cases presented p16(INK4a) mRNA overexpression (p = 0.03). Our findings suggest that p16(INK4a) overexpression, both at protein and mRNA levels, may reflect CDKN2A genetic alterations in HPV-negative laryngeal squamous cell carcinomas.