Polyembryony, which is a specific true-seed propagation system generated by somatic embryogenesis in nucellar tissue, occurs widely in the genus Citrus. To refine the genetic map of the polyembryony locus in Citrus, we obtained DNA markers from bulked segregation analysis using an F 1 population derived from 'Kiyomi' ('Miyagawa wase' (Citrus unshiu Marcow) × 'Trovita' orange (C. sinensis (L.) Osbeck)) × 'Miyagawa wase'. A total of 1681 primers designed from expressed sequence tag (EST) sequences and random 12-base primers were utilized for bulked segregation analysis with modification to screen markers systematically. Five markers linked to the polyembryony locus were obtained, of which two of these markers were converted to sequence characterized amplified region (SCAR) markers. These markers were integrated to a framework map previously constructed by EST-derived cleaved amplified polymorphic sequence (CAPS) markers and placed on the previously reported K-9 linkage group. The refined linkage group with new markers covered 47 cM flanking the polyembryony locus. From haplotype analysis of the region around the polyembyony locus in the progenies and their parents, it was shown that 'Kiyomi' had a recombinant haplotype in the flanking region close to the polyembryony locus. Information concerning the linkage map and haplotype structure contributes to marker-assisted selection and further study of the polyembryony locus in Citrus.