Our study aimed at elucidating which genetic alterations tend to form a network and could be applied as molecular markers of larynx squamous cell carcinoma (LSCC). A panel of genes involved in tumorigenesis was investigated. To search for the possible mechanisms of gene silencing, loss of heterozygosity (LOH) was analysed followed by testing DNA methylation and protein expression for those genes found with the highest frequency of LOH (CDKN2A (55.4%), MLH1 (46.0%), RB1 (35.7%)). A correlation of both LOH and hypermethylation with the loss of expression for CDKN2A and MLH1 was found. Disrupted Rb pathway (loss of expression of RB1 and/or of CDKN2A) in 55.9% of analysed cases confirmed the hypothesis that RB1 pathway is altered in head and neck squamous cell carcinomas, with CDKN2A (45%), rather than RB1 (11.8%) being more frequently inactivated. In LSCC, LOH tends to occur together in gene pairs or triplets. The pair MLH1/CDKN2A and triplets MLH1/TSG on 8p22/CDKN2A and MLH1/CDKN2A/RB1 are related to staging and grading. LOH in MLH1 correlates with lower and LOH in CDKN2A with higher grades of LSCC. It can be concluded that MLH1 and CDKN2A play an important role in LSCC development and progression.