The tyrosinase (TYR) gene is the major melanogenesis-related gene for skin (fur) or plumage color in mammals and birds. Genetic variation in the promoter region of a gene may affect gene expression and phenotypes. This study compared the TYR promoter region between pooled DNA (n = 8) of chickens (Gallus gallus) with black and white skin using direct sequencing. The single nucleotide polymorphism (SNP) c.-2228A>T was found to have the opposite allele distribution in the two groups. The results of genotyping in a larger population (n = 188) revealed that SNP c.-2228A>T was associated with the skin color of the black-boned chicken. Individuals with genotypes AA and AT had greater TYR expression than those with genotype TT. A luciferase assay of the promoter activity revealed that genotype AA had greater activity than genotype TT. Transcription factor binding site analyses showed that the c.-2228A allele has a putative binding site for transcription factor AT-rich interaction domain 3a (Arid3a), while the c.-2228T allele has sites for GS homeobox 2 (GSX2), homeobox D9 (Hoxd9), and mix paired-like homeobox (MIXL1). We concluded that the TYR promoter polymorphism affects skin color. SNP c.-2228A>T could be used as a genetic marker for marker-assisted selection of skin color in the black-boned chicken.