Background: Penile squamous cell carcinoma (PSCC) is rare, representing 0.4-0.6% of all male malignancies in the global-north, as compared with 2-10% in the global-south. Overall survival (OS) dramatically decreases with increased lymph node metastases (LNM). Increased prevalence of pro-tumorigenic M2 CD163+ tumour associated macrophages (TAM) is associated with poor PSCC OS, whereas increased prevalence of the generic M1/2 CD68+ TAMs is associated with better PSCC OS. PDL-1 expression has been noted in 40-60% of PSCC primary lesions. However, there is a paucity of evidence on the overall tumour immune microenvironment (TME) within penile LNM disease. This study describes the relationship between PDL-1 expression and TAMs within the TME of the primary tumour and paired LNMs, with the goal of predicting the utility of PD-1 inhibitors in clinical trials in PSCC. Methods: The Ventana Discovery ULTRA platform was used to optimise a 7-marker multiplex immunofluorescence panel on whole FFPE sections of primary and LNM lesions. Multispectral images were captured using a Vectra 3 microscope. Single cell spatial analysis was performed using HALO software, with statistical analysis of data using R 4.2.1. Results: 52 cases with differing LN stages were identified: N0 (N=20); N1/N2 (N=21); N3 (N=11). Increased expression of PDL-1 was noted at the primary lesion invasive margin (IM) of N+, compared with N0 patients. High expression in inguinal LNMs (iLN+) and pelvic LNMs (pLN+) was also observed, with no expression detected in microscopically normal, tumour free LNs (LN-) in the N0 cohort. More CD163+ M2 TAMs were observed at the IM of N+ compared with N0. However, no significant difference was observed between the three LN groups. M1/2 CD68+ TAMs were more prevalent within pLN+ vs iLN+ or LN-. There was no significant difference in M1/2 CD68+ TAMs number at the IM. Conclusions: Our data highlights increased prevalence of M2 CD163+ TAMs within the IM is associated with LNM. Increased presence of CD68+ M1/2 TAMs in pLN+ may represent active inflammation at the leading microscopic edge of metastatic deposits. A significant difference in the expression of PDL-1 between localised and metastatic disease at both primary and LN location is noted. High PDL-1 expression at the IM of the primary lesion is associated with increased LNMs, where PDL-1 expression is significantly higher as compared to LN-. This study supports the rationale for the use of anti-PD-1/PDL-1 therapies in patients with LNM disease.