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Abstract Background Human papillomavirus (HPV) is a causative agent for tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC), as well as for cervical cancer. Premalignant stages in cervical cancer have been studied extensively, while little is known about premalignant stages in TSCC/BOTSCC and the role of HPV. Here we analyzed differences in gene and protein expression between high‐grade dysplasia and invasive cancer in both HPV‐positive (HPV+) and HPV‐negative (HPV−) TSCC/BOTSCC. Methods High‐grade dysplasia and invasive carcinoma were laser microdissected from HPV+ and HPV− TSCC/BOTSCC tumor sections. Differential gene expression was studied utilizing nanoString RNA‐panels and genes of interest were validated on the protein level by immunohistochemistry. Results Forty genes in the HPV+ tumors showed significantly different expression between high‐grade dysplasia and invasive cancer and 33 genes in the HPV− tumors. Five out of the nine most significant pathways showed similar increased activity in invasive cancer as compared to high‐grade dysplasia in both HPV+ and HPV− tumors. Lastly, significant differences in protein expression was confirmed for SPARC, psoriasin, type I collagen and galectin‐1 in both HPV+ and HPV− tumors. Conclusions This is to our knowledge the first study disclosing differences and similarities in gene expression between dysplastic and invasive HPV+ and HPV− TSCC/BOTSCC.