ABSTRACT Chromosome sizes of 71 phytoplasmas belonging to 12 major phylogenetic groups including several of the aster yellows subgroups were estimated from electrophoretic mobilities of full-length chromosomes in pulsed-field gels. Considerable variation in genome size, from 660 to 1,130 kilobases (kb), was observed among aster yellows phytoplasmas. Chromosome size heterogeneity was also observed in the stolbur phytoplasma group (range 860 to 1,350 kb); in this group, isolate STOLF contains the largest chromosome found in a phytoplasma to date. A wide range of chromosome sizes, from 670 to 1,075 kb, was also identified in the X-disease group. The other phytoplasmas examined, which included members of the apple proliferation, Italian alfalfa witches' broom, faba bean phyllody, pigeon pea witches' broom, sugarcane white leaf, Bermuda grass white leaf, ash yellows, clover proliferation, and elm yellows groups, all have chromosomes smaller than 1 megabase, and the size ranges within each of these groups is narrower than in the aster yellows, stolbur, and X-disease groups. The smallest chromosome, approximately 530 kb, was found in two Bermuda grass white leaf phytoplasma isolates. This not only is the smallest mollicute chromosome found to date, but also is the smallest chromosome known for any cell. More than one large DNA band was observed in several phytoplasma preparations. Possible explanations for the occurrence of more than one band may be infection of the host plant by different phytoplasmas, the presence of more than one chromosome in the same organism, or the presence of large extrachromosomal DNA elements.