Cytoplasmic male sterility (CMS) in commercial sunflower hybrids is thought to be derived from a related wild species, Helianthus petiolaris, yet CMS lines are known to carry the chloroplast DNA genotype of H. annuus. To clarify the origin of sunflower CMS, we developed a polymerase chain reaction-based strategy for detecting CMS in sunflower and surveyed more than 1,200 plants representing 55 accessions of H. annuus and 26 accessions of H. petiolaris. We also tested 160 progeny from three crosses for strict maternal inheritance of organelle DNAs to determine if the apparent discrepancy in the species donor of the mitochondrial DNA and chloroplast DNA genotypes in CMS lines might result from low-frequency maternal or biparental inheritance of either organelle. No CMS cytotypes were observed in natural populations of either H. annuus or H. petiolaris, and strict maternal inheritance of organelle DNA was observed. These data provide little insight, therefore, into the origin and population genetics of CMS in natural populations of sunflower, except that the evidence for strict maternal inheritance of organelles in sunflower makes it unlikely that the mtDNA and cpDNA genotypes in CMS lines were derived from different species. Nonetheless, the primers developed for assaying organelle DNA variation in sunflower may be useful tools for plant breeding programs, cytotype identification, and systematic and evolutionary studies in the domesticated sunflower and its relatives.