Melanomas originating from mucosal surfaces have low mutation burden, genomic instability, and poor prognosis. To identify potential driver genes, we sequenced hundreds of cancer-related genes in 43 human mucosal melanomas, cataloging point mutations, amplifications, and deletions. The gene, which encodes a negative regulator of mitogen-activated protein kinase (MAPK) signaling, was inactivated in 37% of the tumors. Four distinct genotypes were associated with loss. Using a rapid, tissue-specific CRISPR technique to model these genotypes in zebrafish, we found that functions as a tumor suppressor, particularly in the context of mutations. knockdown caused MAPK activation, increased cell proliferation, and conferred resistance to drugs inhibiting KIT tyrosine kinase activity. These findings provide a rationale for MAPK inhibition in SPRED1-deficient melanomas and introduce a zebrafish modeling approach that can be used more generally to dissect genetic interactions in cancer. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.