The red imported fire ants, Solenopsis invicta Buren are one of the serious pests in the world. Recently, S. invicta and S. germinata were found at Gamman pier, Pusan port in Korea by Animal and Plant Quarantine Agency. It is difficult to discriminate between S. invicta and S. germinata because of their morphology. Although DNA barcoding is an efficient method for species identification based on partial sequence of the mitochondrial cytochrome oxidase I gene(COI), amplification of non-target sequences or heteroplasmic COI sequences may result in the failure of Sanger sequencing. To overcome these limitations, application of next generation sequencing platforms is an alternative method. Here, we developed SNP markers using NGS technologies to distinguish two fire ants accurately and easily. For read mapping from high-throughput sequencing data, we used S. invicta reference genome version Si_gnG downloaded from NCBI database. Samtools were used for genotypes calling after mapping read to reference genome by BWA. After coding sequence SNPs that clearly distinguish S. invicta and S. geminata from one candidate gene were selected, the SNPs were validated additional Sanger sequencing using various accessions, including S. invicta and S. geminata. Our results demonstrated that detection of S. invicta from Solenopsis genus group could be accurately and quickly detected by these markers.