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The cytogenetic characterization of the carrot genome (Daucus carota L., 2n = 18) has been limited so far, partly because of its somatic chromosome morphology and scant of chromosome markers. Here, we integrate the carrot linkage groups with pachytene chromosomes by fluorescent in situ hybridization (FISH) mapping genetically anchored bacterial artificial chromosomes (BACs). We isolated a satellite repeat from the centromeric regions of the carrot chromosomes, which facilitated the study of the pachytene-based karyotype and demonstrated that heterochromatic domains were mainly confined to the pericentromeric regions of each chromosome. Chromosome-specific BACs were used to: (1) physically locate genetically unanchored DNA sequences, (2) reveal relationships between genetic and physical distances, and (3) address chromosome evolution in Daucus. Most carrot BACs generated distinct FISH signals in 22-chromosome Daucus species, providing evidence for syntenic chromosome segments and rearrangements among them. These results provide a foundation for further cytogenetic characterization and chromosome evolution studies in Daucus.