The coloration of plumage in poultry species has substantial economic significance. Putian black ducks encompass 2 distinct strains characterized by black and white plumage variations resulting from selective breeding. This study aimed to identify the molecular mechanisms responsible for plumage coloration in these 2 distinct strains. A comprehensive genome-wide association study was conducted using DNA data sourced from a F2 segregating population, consisting of 71 individuals with black plumage and 39 individuals with white plumage, derived from these distinct 2 strains. This analysis revealed that 894 nucleotide polymorphisms and identified 58 candidate genes. Subsequent Gene Ontology and Kyoto Encyclopedia of Genes and Genomes coenrichment analyses identified MITF as a key candidate gene implicated in melanin biosynthesis. Furthermore, extensive screening of significant polymorphic loci within MITF was carried out via mass spectrometry in 3 distinct populations: 100 individuals with black plumage and 100 individuals with white plumage from the F0 generation; and 50 with black plumage form the F1 generation). Eighteen candidate polymorphic loci were identified demonstrating significant associations with variations in black and white plumage. Notably, 8 of these loci were located within the 2,000 bp region upstream of MITF-M. To validate the critical regulatory role of MITF-M in black and white plumage formation, a dual-fluorescence reporter system was constructed, and dual-fluorescence activity was assessed. The results revealed that the fluorescence activity at wild-type sites (corresponding to black plumage) was significantly higher than that at the mutant-type sites (associated with white plumage) (P < 0.01). To corroborate the pivotal role of MITF-M in black and white plumage formation, qPCR was employed to evaluate the expression levels of various MITF variants in black and white feather bulbs. This analysis demonstrated that only MITF-M exhibited specific expression in black feather bulbs. These results elucidate the central role of polymorphic mutations within the MITF promoter region in the regulation of black and white plumage coloration in Putian black ducks. This study extends our understanding of mechanisms governing duck plumage coloration and provides valuable molecular markers for future research in duck production and breeding based on plumage coloration. All rights reserved, Elsevier.