This study examined PRAME expression by immunohistochemistry and reverse transcription quantitative PCR (RT-qPCR) in 202 histologically unequivocal conjunctival melanocytic lesions: 76 nevi, 29 benign melanoses, 25 low-grade conjunctival intraepithelial melanocytic lesions (LGCMIL), 26 high-grade conjunctival melanocytic intraepithelial lesions/in-situ melanoma (HGCMIL) and 46 invasive melanomas. PRAME score 0 was seen in 96% of nevi (73/76), 96% of benign melanoses (28/29) and 88% of LGCMIL (22/25). PRAME score 4 was seen in 50% HGCMIL (13/26) and 76% invasive melanomas (35/46). PRAME score 4 had sensitivity of 50% and specificity of 100% in differentiating between HGCMIL and benign melanosis/LGCMIL. PRAME score 4 had sensitivity of 76% and specificity of 100% in differentiating between melanoma and nevi. Relative quantification of PRAME mRNA expression by RT-qPCR was performed on 49 (24%) cases: 17 nevi, 3 benign melanoses, 5 LGCMIL, 9 HGCMIL, and 15 invasive melanomas. The analysis generated two distinct groupings with 'high' relative PRAME expression for the HGCMIL and invasive melanoma and 'low/zero' expression for nevi, benign melanosis and LGCMIL. Thirty-three challenging conjunctival melanocytic lesions that had previous FISH analysis were studied: 18 nevi, 12 melanomas in a nevus, 2 nevoid melanomas, 1 in-situ melanoma. All nevi (100%) showed concordance between negative FISH and PRAME (scores 0-3). 4/13 (31%) melanomas (in-situ, invasive, isolated, and in association with nevus) showed concordance between positive FISH and PRAME score 4. In conclusion, PRAME score 4 has 100% specificity for diagnosis of HGCMIL and melanoma. PRAME is limited in its sensitivity in the evaluation of challenging melanocytic lesions.