Importance The main high-penetrance melanoma susceptibility gene is CDKN2A, encoding p16INK4A and p14ARF. The gene MITF variant p.E318K also predisposes to melanoma and renal cell carcinoma. To date, the prevalence of MITF p.E318K and its clinical and phenotypical implications has not been previously assessed in a single cohort of Spanish patients with melanoma or in p16INK4A mutation carriers.Objectives To evaluate the prevalence of MITF p.E318K in Spanish patients with melanoma and assess the association with clinical and phenotypic features.Design, Setting, and Participants A hospital-based, case-control study was conducted at the Melanoma Unit of Hospital Clinic of Barcelona, with MITF p.E318K genotyped in all patients using TaqMan probes. We included 531 patients: 271 patients with multiple primary melanoma (MPM) without mutations affecting p16INK4A (wild-type p16INK4A); 191 probands from melanoma-prone families with a single melanoma diagnosis and without mutations affecting p16INK4A, and 69 probands from different families carrying CDKN2A mutations affecting p16INK4A. A population-based series of 499 age- and sex-matched cancer-free individuals from the Spanish National Bank of DNA were included as controls. Patients were recruited between January 1, 1992, and June 30, 2014; data analysis was conducted from September 1 to November 30, 2014.Main Outcomes and Measures The genetic results of the MITF p.E318K variant were correlated with clinical and phenotypic features.Results Among the 531 patients, the prevalence of the MITF p.E318K variant was calculated among the different subsets of patients included and was 1.9% (9 of 462) in all melanoma patients with wild-type p16INK4A, 2.6% (7 of 271) in those with MPM, and 2.9% (2 of 69) in the probands of families with p16INK4A mutations. With results reported as odds ratio (95% CI), the MITF p.E318K was associated with an increased melanoma risk (3.3 [1.43-7.43]; P < .01), especially in MPM (4.5 [1.83-11.01]; P < .01) and high nevi count (>200 nevi) (8.4 [2.14-33.19]; P < .01). Two fast-growing melanomas were detected among 2 MITF p.E318K carriers during dermatologic digital follow-up.Conclusions and Relevance In addition to melanoma risk, MITF p.E318K is associated with a high nevi count and could play a role in fast-growing melanomas. Testing for MITF p.E318K should not exclude patients with known mutations in p16INK4A. Strict dermatologic surveillance, periodic self-examination, and renal cell carcinoma surveillance should be encouraged in this context.