KEY MESSAGE: The sugar beet Rf1 locus has a number of molecular variants. We found that one of the molecular variants is a weak allele of a previously identified allele. Male sterility (MS) caused by nuclear-mitochondrial interaction is called cytoplasmic male sterility (CMS) in which MS-inducing mitochondria are suppressed by a nuclear gene, restorer-of-fertility. Rf and rf are the suppressing and non-suppressing alleles, respectively. This dichotomic view, however, seems somewhat unsatisfactory to explain the recently discovered molecular diversity of Rf loci. In the present study, we first identified sugar beet line NK-305 as a new source of Rf1. Our crossing experiment revealed that NK-305 Rf1 is likely a semi-dominant allele that restores partial fertility when heterozygous but full fertility when homozygous, whereas Rf1 from another sugar beet line appeared to be a dominant allele. Proper degeneration of anther tapetum is a prerequisite for pollen development; thus, we compared tapetal degeneration in the NK-305 Rf1 heterozygote and the homozygote. Degeneration occurred in both genotypes but to a lesser extent in the heterozygote, suggesting an association between NK-305 Rf1 dose and incompleteness of tapetal degeneration leading to partial fertility. Our protein analyses revealed a quantitative correlation between NK-305 Rf1 dose and a reduction in the accumulation of a 250 kDa mitochondrial protein complex consisting of a CMS-specific mitochondrial protein encoded by MS-inducing mitochondria. The abundance of Rf1 transcripts correlated with NK-305 Rf1 dose. The molecular organization of NK-305 Rf1 suggested that this allele evolved through intergenic recombination. We propose that the sugar beet Rf1 locus has a series of multiple alleles that differ in their ability to restore fertility and are reflective of the complexity of Rf evolution.