The mitochondrial (mt) genomes from D₂ (Gossypium harknessii) male sterile-inducing cytoplasm and normal upland cotton AD₁ (G. hirsutum) cytoplasm were compared by restriction fragment length polymorphism (RFLP) analysis. In the RFLP analysis, seven heterologous probes and 5 restriction enzymes were used. The results showed considerable differences in the overall mt genomic structure between the two cytoplasms. With coxI, coxII, atp6, atp9, and atpA as probes, minor differences in fragment size were detected with one or two restriction enzyme digestions. The most notable difference was an additional fragment in normal cytoplasm detected by coxII in digests of four enzymes, and by coxI and atpA in digests with PstI. However, no difference in RFLP patterns was noted between male sterile (A) and restorer (R) lines with the D₂ cytoplasm, indicating that the presence of the D2 restorer gene does not affect mtDNA organization.