Mitochondrial (mt) DNA variation for six petaloid cytoplasmic male-sterile (CMS) and three fertile maintainer lines of carrot was assessed to establish genetic relationships. Total DNA was digested with restriction enzymes and probed with six homologous mtDNA cosmid probes. The six CMS accessions derived from wild carrot, four from Guelph, Ontario, one from Orleans, Massachusetts, and one from Madison, Wisconsin? were more closely related with each other (F = 0.91) than with fertile maintainer lines derived from cultivated germplasm (F = 0.62). The fertile maintainer lines were likewise found to be more similar to each other (F = 0.78) than to the sterile lines. Three sterile lines, originating from wild carrot populations within 1 km of each other in Guelph, Ontario, were most closely related (F = 0.96). The high degree of similarity among the six petaloid CMS lines which originated from individual wild carrot plants, some from geographically diverse regions, suggests that the cytoplasm responsible for this trait was imported to, or else evolved, only once in North America.