AbstractBy means of gene disruption analyses, Saccharomyces cerevisiae strain YPH499 was shown to have two, and only two, copies of ATP3 that encodes the γ‐subunit of H+‐dependent ATP synthase and locates on the right arm of chromosome II. Linkage analyses of the two distinguishably marked copies of ATP3 indicated that the distance between them was about 43 cM. Since YBR030W, an ORF proximal to ATP3 by a distance of 17 kbp, was also found to be duplicated, we marked them with two distinguishable nutritional markers, which were also distinguishable from those used for marking the two copies of ATP3, and achieved four‐point linkage analyses; CEN2 marked with an appropriate nutritional marker gene was included as a reference point. And, the following linkage map was deduced: CEN2– [11 cM]–YBR030Wa– [8 cM]–ATP3a–[47 cM]–ATP3b– [55 cM]–YBR030Wb. From this map, we suspected that a segment spanning at least YBR030W–ATP3 would be inversely duplicated on the right arm of chromosome II. We then carried out chromosome fragmentation analyses, using several laboratory strains including YPH499, and obtained data in accord with our speculation for all strains, although the distance between the two copies of ATP3 varied from 48 kbp to 192 kbp among the strains examined. Copyright © 2005 John Wiley & Sons, Ltd.