Melting curve analysis of fluorescently labeled DNA fragments is used extensively for genotyping single nucleotide polymorphism (SNP). Here, we tested a SNP genotyping method by melting curve analysis with the two probe chemistries on a Roche LightCycler 480. The HybProbe chemistry is based on the fluorescence resonance energy transfer (FRET) and the SimpleProbe chemistry uses a terminal self-quenching fluorophore. We evaluated FRET HybProbes and SimpleProbes for two SNP sites closely linked to southern root-knot nematode resistance. These probes were used to genotype the two parents and 94 F_2:3 populations from the cross of PI 96354 x Bossier. The genotypes of all samples determined by the LightCycler software agreed with previously determined SSR and Luminex flow cytometry data. Multiplexed HybProbes showed a 98.4% successful rate and 100% concordance between two replicates. The LightCycler provides rapid PCR on 384-well plate and allows simultaneous amplification and analysis in approximately 2 hours without any additional steps after amplification. This allowed for a reduction of the potential contamination of PCR products, simplicity, and enablement of a streamlined workflow. The melting curve analysis on the LightCycler provided high-throughput and rapid SNP genotyping and was highly desirable for marker-assisted selection in soybean.