Background: Biomarkers are needed in primary melanoma to risk stratify for adjuvant trials. High levels of infiltrating cytotoxic (CD8+) T lymphocytes (CTLs) and low levels of CD68+ macrophages (MΦ) may correlate with prolonged survival but quantification methods are not standardized for clinical practice. HLA-DR is a marker of MΦ activation not expressed by suppressor myeloid cells. A novel pathology technique using mIHC allows for quantitative and spatial analysis of immune cell subsets. Methods: In a pilot set of stage II/III primary melanomas from Columbia University Medical Center (n = 94), clinical follow up is available for 51 cases. 32 had no evidence of recurrence at last follow up (minimum 2 years) while 19 died of melanoma. 5µm slides were stained using Opal multiplexed IHC (mIHC) for DAPI, CD3, CD8, CD68, SOX10, HLA-DR and Ki67. Tumor areas were pre-selected by a dermatopathologist, visualized using Mantra (Perkin Elmer) and analyzed using inForm (Perkin Elmer) and Spotfire (TIBCO). Results: In all patients (n = 94), CTLs are farther from tumor (SOX10+) cells when they are proliferating (Ki67+) (p < 0.0001***), while they are closer to MΦ when they are activated (HLA-DR+) (p = 0.0002***). Next, we evaluated impact on prognosis using disease specific survival (DSS) as an outcome based on median value (n = 51). In this exploratory study no correction for multiple comparisons was made. We find that CTL density correlates with prolonged DSS in tumor (p = 0.0185*) but not in stroma (p = 0.1630 ns). Ratio of density of CD8/CD68+HLA-DR- correlates with DSS in both tumor (p = 0.027*) and stroma (p = 0.017*). Finally, distance from CTLs to HLA-DR- MΦ was significantly greater in non-recurrent melanomas as compared to recurrent ones (p = 0.0167*). Conclusions: HLA-DR expression on MΦ and Ki67 expression on tumor cells correlate with position of CTLs in TME in primary melanoma. CTL density is a favorable prognostic marker while HLA-DR non-expressing MΦ may favor tumor progression. Quantitative mIHC allows for accurate spatial analysis of immune subsets within the TME and the development of novel, more accurate and potentially clinically relevant biomarkers.