Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in resource poor countries where the majority of tuberculosis cases occur.Spoligotyping is a PCR-based Mycobacterium tuberculosis complex genotyping method with advantages of technical simplicity, numerical output and high reproducibility. It is based on presence or absence of 43 distinct « spacers » separating insertion element in the direct repeat region of the Mycobacterium tuberculosis genome. Spoligotyping assay involves reverse hybridization of PCR products to the capture spacers attached to nitrocellulose membranes or to microspheres. Here we report modification of the classic 43-spacer method using the new generation of Luminex multiplexing technology with magnetic microspheres.The method was successfully established and validated on strains with known spoligotypes in our laboratory in Haiti. Distribution of spoligotypes determined in a collection of 764 recent Mycobacterium tuberculosis isolates was in accordance with previous data for Haitian isolates in the SITWITWEB international database, which were obtained with traditional membrane-based method. In the present form, spoligotyping may be suitable as a high-throughput, first-line tool for genotyping of Mycobacterium tuberculosis in countries with limited resources.