This study aimed to utilize next-generation sequencing for developing microsatellite markers in Fulvia mutica. The Illumina NovaSeq 6000 System generated a total of 327,584,690 reads, resulting in a sequence length of 49,465,288,190 bp. The de novo assembly yielded an 885 Mb (885,020,821 bp) sequence. In this context, 675,057 contigs longer than 500 bp were identified. Among these, 107,985 contigs containing microsatellite regions were identified, accounting for 15.9%. A total of 105,632 microsatellite loci were found within these 107,985 microsatellite regions. Among these loci, polymorphism information was confirmed at 3,723 (3.5%) microsatellite marker positions. eighty-two primer sets were designed based on the 150 microsatellite loci. As a result, 14 microsatellite loci were selected for estimating population genetic parameters in both wild populations. The mean number of effective alleles was 11, ranging from 7 to 18. The observed heterozygosity (HO) ranged from 0.615 to 0.937, with an average of 0.790, while the expected heterozygosity (HE) ranged from 0.629 to 0.924, also with an average of 0.790. Based on these findings, the compiled panel of 14 microsatellite markers is anticipated to facilitate examining genetic traits within the Fulvia mutica population in Korea. The results of the analysis of genetic diversity and gene relationships in Fulvia mutica are expected to be crucial data for the management, conservation, and sustainable utilization of genetic resources. Such information is expected to play a pivotal role in developing conservation and management strategies and formulating policies and strategies for sustainable utilization of genetic resources.