Background <p>Recently, the heterogeneity of human papillomavirus (HPV) status within HPV-associated oropharyngeal squamous cell carcinoma (HPV-OPSCC) has garnered substantial attention. However, the origin and relevance of a subset of cancer cells with HPV-loss remain largely unknown.</p> Methods <p>We encountered seven HPV-OPSCCs with spatial heterogeneity identified by HPV-DNA in situ hybridization among 83 OPSCCs, including 48 HPV-OPSCCs. We analyzed the characteristics of HPV-loss and HPV-positive cancer cells using histomorphology and immunohistochemistry. Spatial transcriptomic profiling was performed to investigate the evolutionary dynamics and pathway alterations associated with the emergence of HPV-loss tumor cells, as well as the corresponding changes in the tumor microenvironment. Additionally, we evaluated the clinical prognosis of OPSCCs exhibiting intratumoral HPV-DNA heterogeneity.</p> Results <p>We demonstrated that HPV-loss subsets originate from HPV-positive cancer cells and diverge into distinct lineages during spatiotemporal evolution. This transition was associated with hypoxia and PI3K/AKT/mTOR signaling. Although HPV-loss clones showed more aggressive features such as poor differentiation and strong p16 expression, the overall prognosis of patients with HPV-DNA spatial heterogeneity was comparable to those with homogeneous HPV status. This may be due to increased interferon signaling and elevated cytotoxic lymphocyte infiltration in HPV-loss clones, suggesting a loss of immune evasion. In contrast, HPV-positive clones retained immune evasion mechanisms, potentially mediated by Fibroblast Growth Factor Receptor 3 signaling.</p> Conclusions <p>We identified the emergence of HPV-loss clones in HPV-OPSCC and uncovered their spatiotemporal evolution characteristics, as well as their impact on the tumor microenvironment.</p>