Gross variations in the structure of the centromere of Schizosaccharomyces pombe chromosome III (cen3) were apparent following characterization of this centromeric DNA in strain Sp223 and comparison of the structure with that of cen3 in three other commonly used laboratory strains. Further differences in centromere structure were revealed when the structure of the centromere of S. pombe chromosome II (cen2) was compared among common laboratory strains and when the structures of cen2 and cen3 from our laboratory strains were compared with those reported from other laboratories. Differences observed in cen3 structure include variations in the arrangement of the centromeric K repeats and an inverted orientation of the conserved centromeric central core. In addition, we have identified two laboratory strains that contain a minimal cen2 repeat structure that lacks the tandem copies of the cen2-specific block of K-L-B-J repeats characteristic of Sp223 cen2. We have also determined that certain centromeric DNA structural motifs are relatively conserved among the four laboratory strains and eight additional wild-type S. pombe strains isolated from various food and beverage sources. We conclude that in S. pombe, as in higher eukaryotes, the centromere of a particular chromosome is not a defined genetic locus but can contain significant variability. However, the basic DNA structural motif of a central core immediately flanked by inverted repeats is a common parameter of the S. pombe centromere.