The aim of the investigation was to develop a definitive typing system for Rodentibacter pneumotropicus. A total of 79 strains including the type strain of R. pneumotropicus, all associated with rodents were used to develop a multi-locus sequence typing scheme (MLST). Primers were designed for conserved regions of seven house-keeping genes (atpG, frdB, gdh, pgi, pmi, recA, zwf) and internal fragments of 399–839 bp were sequenced for all strains. The genes were also extracted in full length from whole genomic sequences of 14 strains of which 10 were sequenced in the current study. The number of alleles at the different loci ranged from 5 to 7 and a total of 20 allelic profiles or sequence types were recognized amongst the 79 strains. Analysis of the MLST data showed that some STs have been stable over many years probably circulating in the same colonies and probably transferred between colonies. We assume that this MLST scheme may provide a high level of resolution and might be an excellent tool for studying the population structure and epidemiology of R. pneumotropicus. Further development of the scheme is expected by including more genes and more strains and involve whole genomic sequencing.