Abstract Background Population-wide screening for melanoma is not cost-effective, but genetic characterisation could facilitate risk stratification and targeted screening. Common MC1R red hair colour (RHC) variants and MITF E318K separately confer moderate melanoma susceptibility, but their interactive effects are relatively unexplored. Objectives Evaluate whether MC1R genotypes differentially affect melanoma risk in MITF E318K+ versus E318K- individuals. Methods Melanoma affection status and genotype data (MC1R and MITF E318K) were collated from research cohorts (five Australian and two European). In addition, RHC genotypes from E318K+ individuals with and without melanoma were extracted from databases (The Cancer Genome Atlas and Medical Genome Research Bank respectively). Chi-square and logistic regression evaluated RHC allele and genotype frequencies within E318K+/- cohorts depending on melanoma status. Replication analysis was conducted on 200,000 general population exomes (UK Biobank). Results The cohort comprised of 1,165 MITF E318K- and 322 E318K+ individuals. In E318K- cases MC1R R and r alleles increased melanoma risk relative to wild-type (wt), p<0.001 for both. Similarly, each MC1R RHC genotype (R/R, R/r, R/wt, r/r and r/wt) increased melanoma risk relative to wt/wt (p<0.001 for all). In E318K+ cases, R alleles increased melanoma risk relative to wt allele (OR=2.04, 95% CI [1.67, 2.49], p=0.01), while the r allele risk was comparable to wt allele (OR=0.78, 95% CI [0.54,1.14] vs 1.00 respectively). E318K+ cases with the r/r genotype had a lower but not significant melanoma risk relative to wt/wt (OR=0.52, 95% CI [0.20,1.38]). Within the E318K+ cohort, R genotypes (R/R, R/r, and R/wt) conferred a significantly higher risk compared to non-R genotypes (r/r, r/wt and wt/wt) (p<0.001). UK Biobank data supporting our findings that r did not increase melanoma risk in E318K+ individuals. Conclusions RHC alleles/genotypes modify melanoma risk differently in MITF E318K- and E318K+ individuals. Specifically, although all RHC alleles increase risk relative to wt in E318K- individuals, only MC1R R increases melanoma risk in E318K+ individuals. Importantly, in the E318K+ cohort the MC1R r allele risk is comparable to wt. These findings could inform counselling and management for MITF E318K+ individuals.