AbstractThe management of anal squamous cell carcinoma (ASCC) has yet to experience the transformative impact of precision medicine. Conducting genomic analyses may uncover novel prognostic biomarkers and offer potential directions for the development of targeted therapies. To that end, we assessed the prognostic and theragnostic implications of pathogenic variants identified in 571 cancer‐related genes from surgical samples collected from a homogeneous, multicentric French cohort of 158 ASCC patients who underwent abdominoperineal resection treatment. Alterations in PI3K/AKT/mTOR, chromatin remodeling, and Notch pathways were frequent in HPV‐positive tumors, while HPV‐negative tumors often harbored variants in cell cycle regulation and genome integrity maintenance genes (e.g., frequent TP53 and TERT promoter mutations). In patients with HPV‐positive tumors, KMT2C and PIK3CA exon 9/20 pathogenic variants were associated with worse overall survival in multivariate analysis (Hazard ratio (HR)_KMT2C = 2.54, 95%CI = [1.25,5.17], P value = .010; HR_PIK3CA = 2.43, 95%CI = [1.3,4.56], P value = .006). Alterations with theragnostic value in another cancer type was detected in 43% of patients. These results suggest that PIK3CA and KMT2C pathogenic variants are independent prognostic factors in patients with ASCC with HPV‐positive tumors treated by abdominoperineal resection. And, importantly, the high prevalence of alterations bearing potential theragnostic value strongly supports the use of genomic profiling to allow patient enrollment in precision medicine clinical trials.