Occasional reports indicated cytokeratin (CK) protein expression (mainly by immunohistochemistry) in malignant melanoma (MM) and suggested an association with unfavorable clinical parameters. However, the mRNA expression of CK and its clinicopathologic significance in MM has not been specifically evaluated. We investigated the mRNA and protein expression of nine CKs in melanoma cell lines and tissues, in particular the prognostic significance of CK18 mRNA expression. Reverse transcription (RT)-PCR (CK6–10, 14 and 18–20), in-situ hybridization (ISH) (CK18), and western blotting (CK18 and pan-cytokeratin AE1/AE3) were performed on MM cell lines A375, A875, M14, and SK-MEL-1. Eighty MM tissue samples were analyzed by ISH and immunohistochemistry for CK18 expression. The mRNA of CK6–8, 10, 14, 18, and 19 (but not CK9 and 20) was detected in one to four of the melanoma cell lines by RT-PCR. CK18 was detected in all four cell lines by RT-PCR, ISH, and western blotting. CK18 mRNA ISH was positive in three of 30 (10.0%), 10 of 25 (40.0%), and 12 of 25 (48.0%) of primary cutaneous, primary mucosal, and metastatic melanomas, respectively (overall positivity: 25 of 80, 31.3%). CK18 immunostaining was only observed focally in eight of 80 (10.0%) of MM tissue samples, and AE1/AE3 immunostaining was altogether negative. Significantly, CK18 mRNA ISH positivity (but not protein immunohistochemistry) was associated with poorer prognosis by both univariate analysis (P<0.001) and multivariate analysis (relative risk=5.430, 95% confidence interval 2.246–13.128, P<0.001). CK18 mRNA could be identified in one-third of melanoma tissue samples and is an adverse prognostic factor. ISH is superior to immunohistochemistry for analyzing CK18 expression in MM.