Sugar beet is an economically significant crop, and the homozygosity of paired monogerm cytoplasmic male sterile (CMS) and maintainer lines directly influences the number of hybrid combinations that can be created. This study aimed to evaluate the genetic variation within monogerm sugar beet germplasm resources to establish a foundation for advancements in sugar beet breeding and the development of hybrid female parent lines. This study analyzed the genetic diversity of 86 distinct monogerm germplasm resources, including 38 paired monogerm CMS and maintainer lines, 5 individual maintainer lines, and 5 externally introduced sterile lines. The analysis employed 26 pairs of SSR primers and 35 pairs of InDel primers across nine sugar beet chromosomes. Several genetic parameters, and analyses such as structural analysis, genetic diversity analysis, and principal coordinate analysis, were used to evaluate the samples. The results indicated that these strains could be classified into two groups: Group I and Group II. Group I was further divided into three subgroups. Further, 18 pairs of original CMS and maintainer lines were successfully clustered, confirming that their nuclei had achieved homozygosity, making them suitable for use in the development of binary sterile lines. However, 20 other pairs still require further backcrossing to achieve homozygosity. The analysis of molecular variance (AMOVA) revealed that most of the genetic variation occurred within individuals, with relatively low genetic differentiation between groups. Significant genetic differentiation was observed between Subgroups 2 and 3, and between Subgroups 1 and 3. The results suggest that additional monogerm sterile and maintainer lines from these subgroups should be selected to configure binary sterile lines. This study offers a theoretical foundation for developing new sugar beet germplasm resources and cultivating hybrid mother plants.