Abstract Carrot is a major source of provitamin A in a human diet. Two of the most important traits for carrot breeding are carotenoid contents and root color. To examine genomic regions related to these traits and develop DNA markers for carrot breeding, we performed a genome-wide association study (GWAS) using genome-wide single-nucleotide polymorphisms (SNPs) in two F2 populations, both derived from crosses of orange root carrots bred by a Japanese seed company. The GWAS revealed 21 significant associations, and the physical position of some associations suggested two possible candidate genes. An Orange (Or) gene was a possible candidate for visual color evaluation and the α- and β-carotene contents. Sanger sequencing detected a new allele of Or with an SNP which caused a non-synonymous amino acid substitution. Genotypes of this SNP corresponded to the visual evaluation of root color in another breeding line. A chromoplast-specific lycopene β-cyclase (CYC-B) gene was a possible candidate for the β/α carotene ratio. On CYC-B, five amino acid substitutions were detected between parental plants of the F2 population. The detected associations and SNPs on the possible candidate genes will contribute to carrot breeding and the understanding of carotenoid biosynthesis and accumulation in orange carrots.