Sudangrass possesses advantageous traits, including slender stalks and vigorous summer growth. However, its high nutrient absorption efficiency predisposes it to nitrate accumulation in the stems and poses a risk of nitrate poisoning in cattle. This study evaluated 42 lines of 33 sudangrass varieties, comprising germplasm from the National Institute of Agricultural Sciences (NIAS) gene bank and commercial varieties. Sudangrass lines exhibited diverse phenotypic characteristics in the field trials. Genotyping-by-sequencing (GBS) was used to analyze genomic DNA, categorizing sudangrass into three groups based on 3975 single-nucleotide polymorphisms (SNPs). Cluster I, with 22 lines, such as Haysudan, displayed shorter and thicker culms resembling sorghum with fewer tillers. Cluster II, comprising eight lines, including Balesudan, exhibited enhanced tillering and biomass production. Cluster III, represented by 12 lines, such as Piper, showed moderate traits but greater susceptibility to northern leaf blight. Cultivation of sudangrass under varying cattle compost application rates revealed differences in nitrate-nitrogen concentrations, confirming a trend of increased accumulation with higher fertilization levels. Nitrate-nitrogen levels were higher in the first harvest but declined in the second. To meet the regulatory limit of 0.2% total nitrate-nitrogen (dry weight), compost applications below 5 t・10 a⁻¹ are recommended. Among the tested lines, some exhibited nitrate-nitrogen concentrations near the regulatory threshold even under a high compost application rate of 15 t・10 a⁻¹, while maintaining high dry matter yields. Lines such as GB13, which combine high biomass production with low nitrate accumulation, showed great potential as valuable breeding materials.