Technical Abstract: Cucumber plants regenerated from somatic cells occasionally produce progenies with a strong mosaic (MSC) phenotype on cotyledons and leaves. The MSC phenotype shows paternal transmission and the genetic bases of independently derived MSC lines are unknown. Because cucumber mitochondrial genome is paternally transmitted we hybridized 15 cosmids covering the mitochondrial genome of Arabidopsis thaliana to DNA blots of independently derived MSC lines from tissue cultures and wild-type lines. Polymorphism was revealed near the rrn5/rrn18, nad5-exon2, rpl5 coding and JLV5 non-coding regions. Polymorphisms revealed by rrn18 and nad5-exon2 were due to one rearrangement bringing together these two regions. The polymorphism revealed by rpl5 gene was unique to MSC16 and was a rearrangement placing the rpl5 region next to the forward junction of the JLV5 deletion. Five different hybridization patterns were revealed between 17 independently derived tissue culture lines. Passage of cucumber through cell culture may be a unique method to produce novel mitochondrial DNA rearrangements affecting mitochondrial gene expression.