The clarithromycin resistance is the major problem of treatment failure of Helicobacter pylori infection in many countries. The point mutation in 23S rRNA is the main reason and the most common is A-G transitions at position 2143 (A2143G). In the present study, the loop mediated isothermal amplification-restriction fragment length polymorphism (LAMP-RFLP) was developed to detect A2143G mutation in 23S rRNA of H. pylori. The A2143G mutation was examined from 449 positive urease test samples collected between March 2012 and January 2014 using PCR-RFLP method. The LAMP primers were designed to specifically target 23S rRNA of H. pylori and subsequently digested with BsaI. Mutation at A2143G was detected from 10 of 82 (12.2%) glmM gene PCR positive samples. Ladder pattern of DNA bands could be amplified at 65°C within 60 min in the presence of 0.2 mM each of F3 and B3, 1.6 mM each of FIP and BIP, 0.8 mM each of LF and LB, 1.4 mM of deoxynucleoside triphosphates, 0.8 M betaine and 6 mM MgSO4. The ladder DNA bands of wild type and A2143G mutant strains could be distinguished from different sizes of DNA fragments after restriction enzyme analysis. Thus, our LAMP-RFLP assay is a rapid and simple method for the detection of A2143G mutation in 23S rRNA gene in H. pylori which will be useful for primary antimicrobial resistance screening in countries with a high prevalence of clarithromycin resistance.