<p>A total of 116 multigerm sugar beet germplasm resources, which could be used as breeding material and lacked relevant fertility information, were evaluated for their fertility gene composition using different molecular marker techniques. Using variable number tandem repeats (VNTR)—specific primer TR1 identification of male-sterile cytoplasm (S) and normal cytoplasm (N) was conducted. The results showed that 26.76% of the germplasm resources were shown as N-cytoplasm; 24.13% of the germplasm resources were shown as S-cytoplasm; mixed cytoplasmic types accounted for 49.11%. Using cleaved amplified polymorphism sequences (CAPS)—specific primer S17 analysis of nucleus Rf1 loci fertility genes was performed; the results showed that 66 germplasm resources were identified as homozygous 1800&#xa0;bp bands. Enzymatic digestion showed that 53 germplasm resources displayed pattern 5/5, and 13 germplasm resources showed pattern 4/5. Using DNA fragment length polymorphism (DFLP)—specific primer O7 analysis of nucleus Rf2 loci fertility genes was carried out; the results showed that 38 germplasm resources were identified as homozygous 1400&#xa0;bp bands, and two germplasm resources were identified as homozygous 2600&#xa0;bp bands. No sterile line or maintainer line was identified in this identification; the 116 multigerm sugar beet germplasm resources utilized in this experiment can serve as materials for subsequent breeding; initially, the fertility composition of these accessions remained unclear, hindering their effective utilization. Our research has systematically characterized their fertility profiles, enabling targeted selection of specific germplasm for hybrid combinations in the future breeding programs, aiming to provide a reference for the breeding of superior sugar beet varieties.</p>