Background: Macrophage migration inhibitory factor (MIF) might has a role in the development and disease activity of Rheumatoid arthritis (RA). There are several disadvantages in the conventional methods for MIF genotyping and there is a strong need for convenient, flexible, accurate and inexpensive method. Objectives: This study was designed to study the application of single closed tube melting temperature (Tm)-shift method in genotyping of MIF-173 G>C (s755622) gene in RA patients and to compare its result with the conventional PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and to investigate the potential association of these variants of MIF gene with susceptibility and activity of RA. Methodology: GC-rich tail was attached to 5'-end of one of the two allele specific forward primers, such that MIF alleles can be discriminated by the Tm of the PCR products. One hundred RA patients and 40 healthy controls were genotyped for MIF-173 G>C by both Tm-shift method and conventional PCR-RFLP. Results: The comparison of the PCR-RFLP and the Tm-shift method showed one discordant result in 140 samples tested. Retesting this sample with prolonged time of incubation with the restriction enzyme, corrected the result to become 100% agreement between the two methods. No significant association could be found between MIF-173 G>C SNP and risk of RA. C/C genotype is more prone to higher disease activity than other genotypes. Conclusion: The single closed-tube Tm-shift method is reliable, rapid and cost-effective and it is superior to conventional PCR-RFLP in genotyping of MIF.