The stability of cytoplasmic male sterility expression in several genetic backgrounds was investigated in sugar beet (Beta vulgaris L.). Nine genetically heterogenous plants from old cultivars were crossed with a cytoplasmic male-sterile line to obtain 266 F1 plants. Based on marker analysis using a multiallelic DNA marker linked to restorer-of-fertility 1 (Rf1), we divided the F1 plants into 15 genotypes. We evaluated the phenotypes of the F1 plants under two environmental conditions: greenhouse rooms with or without daytime heating during the flowering season. Three phenotypic groups appeared: those consistently expressing male sterility (MS), those consistently having restored pollen fertility, and those expressing MS in a thermo-sensitive manner. All plants in the consistently male-sterile group inherited a specific Rf1 marker type named p4. We tested the potential for thermo-induced male-sterile plants to serve as seed parents for hybrid seed production, and three genotypes were selected. Open pollination by a pollen parental line with a dominant trait of red-pigmented hypocotyls and leaf veins resulted in seed setting on thermo-induced male-sterile plants, indicating that their female organs were functional. More than 99.9% of the progeny expressed the red pigmentation trait; hence, highly pure hybrids were obtained. We determined the nucleotide sequences of Rf1 from the three genotypes: one had a novel allele and two had known alleles, of which one was reported to have been selected previously as a nonrestoring allele at a single US breeding station but not at other stations in the US, or in Europe or Japan, suggesting environmental sensitivity.