Background: Penile Squamous Cell Carcinoma (PSCC) is a rare but often fatal disease. In this study, we characterize the poorly understood immune microenvironment using multiplex immunofluorescence (mIF) and image analysis approaches in 54 patients with PSCC. Methods: Representative blocks of 54 PSCC patients were stained for six immune markers: CD3, CD8, CD68, PD-1, PD-L1, Pancytokeratin and DAPI. Two experienced pathologists using an image analysis system (InForm 2.2.4) divided them into the tumor and stroma compartment and assessed the different densities of cell phenotypes using R studio with results expressed as cells/mm2. The statistical correlations were performed using Fisher’s exact test, Pearson and Log-rank test for Kaplan Meyer plots. Results: 54 patients with confirmed diagnosis of PSCC had a median age of 62 (IQR 50-70). All samples were from the primary penile tumor with the majority of cases being HPV(–) (62%). We observed significantly higher stromal cytotoxic T cells in HPV(+) cases compared to HPV(–) (P=0.04). Using the mean macrophage count as cutoff for positivity, high densities of total tumor macrophages CD68+ were associated with significantly improved estimated median cancer specific survival (CSS) (NA, P=0.04), median overall survival (OS) (68mos vs NA P=0.02) and lower risk of regional recurrence (P=0.04). On the other hand, the high densities of stromal cytotoxic T cells antigen-experienced (CD3+CD8+PD-1+), was associated with significantly worse median OS (27 vs 102mos P=0.05) and median disease free survival (DFS) (18.2mos vs NA P= 0.07). Also, high densities of stromal T cells antigen-experienced (CD3+PDL-1+), were associated with significantly better CSS (NA, P=0.06) and better median OS (142.1 vs 68.8mos P=0.14). Conclusions: Using novel multiplex image analysis to assess the immune microenvironment in primary PSCC, we showed that high macrophage levels were associated with lower risk of recurrence and improved survival outcomes. Moreover, a low level of exhausted stromal cytotoxic PD-1+ T cells was associated with improved PSCC survival. Further characterization of T cell subsets in relation to tumor HPV status is ongoing.