Background: In early stage laryngeal squamous cell carcinoma (LSCC) radiotherapy with curative intent is a major treatment modality. TNM classification is used to define patients eligible for radiotherapy. Studies in early stage glottic LSCC identified several predictive biomarkers associated with local control. However, we recently reported that this predictive value could not be confirmed in supraglottic LSCC. Objective: To examine whether clinical behavior and protein expression patterns of these biomarkers differ between glottic and supraglottic LSCC. Study Design: Retrospective cohort study. Methods: Tumor tissue sections of 196 glottic and 80 supraglottic T1-T2 LSCC treated primarily with RT were assessed immunohistochemically for expression of pAKT, Ki-67 and β-Catenin. Expression data of HIF-1α, CA-IX, OPN, FADD, pFADD, Cyclin D1, Cortactin and EGFR in the same cohort of glottic and supraglottic LSCC, were retrieved from previously reported data. The relationship between glottic and supraglottic sublocalization and clinicopathological, follow-up, and immunohistochemical staining characteristics were evaluated using logistic regression and Cox regression analyses. Results: Glottic LSCC were correlated with male gender (P =.001), hoarseness as a primary symptom (P <.001), T1 tumor stage (P <.001), negative lymph node status (P <.001), and an older age at presentation (P =.004). Supraglottic LSCC patients developed more post-treatment distant metastasis when adjusted for gender, age, and T-status. While supraglottic LSCC was associated with higher expression of HIF-1α (P =.001), Cortactin (P <.001), EGFR (P <.001), and Ki-67 (P =.027), glottic LSCC demonstrated higher expression of CA-IX (P =.005) and Cyclin D1 (P =.001). Conclusion: Differences in clinicopathological and immunohistochemical staining characteristics suggest that T1-T2 glottic and supraglottic LSCC should be considered as different entities. Level of Evidence: N/A. Laryngoscope, 2020.