Aim of this study was to evaluate cellular fatty acid analysis for characterization of spirochetes. Strains were isolated from arthropods collected in South Moravia, Czech Republic. Fatty acid methyl esters (FAME) profile was determined for five Borrelia burgdorferi sensu lato (s.l.) strains isolated from Ixodes ricinus ticks, one "Spironema culicis" strain recovered from mosquito Culex pipiens and seven spirochetal strains (not identified yet) isolated from mosquitoes and blackflies. Analysis was performed using a gas chromatography column in conjunction with Microbial Identification System Sherlock (MIDI Inc., Newark, DE, USA). Results obtained on the basis of cluster analysis of FAME profiles showed, that the B. burgdorferi sensu lato isolates could be well separated from other spirochetal isolates. We recommended method used in this study as a useful tool for preliminary identification of spirochetes isolated from ticks and dipterans.