SummaryWe describe the isolation and characterization of full‐length chromosomes from non‐culturable plant‐pathogenic, mycoplasma‐like organisms (MLOs). MLO chromosomes are circular and their sizes (640 to 1185kbp) are heterogeneous. Divergence in the range of chromosome sizes is apparent between MLOs in the two major MLO disease groups, and chromosome size polymorphism occurs among some related agents. MLO chromosome sizes overlap those of culturable mycoplasmas; consequently, small genome size alone cannot explain MLO non‐culturability. Hybridization with cloned MLO‐specific chromosomal and 16S rRNA probes detected two separate chromosomes in some MLO ‘type’ strains. Large DNA molecules that appear to be MLO megaplasmids were also demonstrated. The ability to characterize full‐length chromosomes from virtually any non‐culturable prokaryote should greatly facilitate the molecular and genetic analysis of these