After 10 years of evaluation in different locations with high levels of incidence of disease, a group of sugarcane somaclones derived from callus tissues was selected for eyespot resistance. Resistance evaluations of four somaclones were performed under field and laboratory conditions. The results confirmed the superiority of two somaclones, one resistant and one tolerant to eyespot disease. Restriction analysis of mitochondrial DN A revealed that the two somaclones had a different DNA organization which distinguished them both from each other and from the donor plant; the restriction profile was similar however to that of the resistant control done. Restriction patterns of a third somaclone, also resistant, were similar to those of the donor plant. Differences among the somaclones were also evident when using a maize ribosomal DNA probe.