The genotyping of Clostridium difficile is generally performed by the analysis of fragment- or amplification-length polymorphism by pulsed field gel electrophoresis (PFGE) or polymerase chain reaction (PCR) ribotyping. However, sequence-based methods allow typing technique standardisation and data comparison. In the present study 100 C. difficile isolates, obtained from various institutions in the state of Saarland, Germany, were prospectively analyzed by surface layer protein A single locus sequence typing (slpAST). A high proportion (52%) of isolates attributable to ribotype 027 (RT027) was found indicating that the new outbreak strain has become endemic, at least in parts of Germany. RT027 strains displayed characteristic mutations of the potential toxin repressor gene tcdC and antibiotic resistance to macrolides and fluoroquinolones. C. difficile isolates attributable to ribotypes RT001 (27%), RT014/066 (5%), RT078 (4%), to the smz genotype (3%), and to more sporadic genotypes were also identified. Overall, the prevalence of strains with resistance to macrolides or fluoroquinolones was >80%. slpAST allows the comprehensive identification of C. difficile strains by global data comparison, exemplified here by our identification of smz strains previously identified by slpAST of a Japanese outbreak. In conclusion, slpAST appears to be a powerful discriminative tool for the straightforward, standardised genotyping of C. difficile isolates.