MultiLocus Sequence Typing (MLST) is a sequence-based methodused to characterize bacterial genomes. This method was usedto examine the genetic structure of Medicago-nodulating rhizobiaat the Amra site, which is located in an arid region of Tunisia.Here the annual medics M. laciniata and M. truncatula are partof the natural flora. The goal of this study was to identifywhether distinct chromosomal groups of rhizobia nodulate M.laciniata because of its restricted requirement for specificrhizobia. The MLST analysis involved the determination of sequencevariation in 10 chromosomal loci of 74 isolates each from M.laciniata and M. truncatula. Medicago truncatula was used asa control trap host, because unlike M. laciniata, it is relativelyunrestricted in its rhizobial requirements. Allelic diversityamong the plasmid-encoded nodC alleles in the isolates was alsodetermined. The 148 isolates were placed into 26 chromosomalSequence Types (STs), of which only 3 had been identified previously.The rhizobia of M. laciniata were shown to be part of the generalMedicago-nodulating population in this soil because 99.95% ofthe isolates had chromosomal genotypes similar to those recoveredfrom M. truncatula. However, the isolates recovered from M.laciniata were less diverse than those from M. truncatula andthey also harbored an unusual nodC allele. This could perhapsbe best explained by horizontal transfer of the different nodCalleles among members of the Medicago-nodulating rhizobial populationat the field site. Evidence is provided indicating a historyof lateral transfer of rhizobial symbiotic genes across distinctchromosomal backgrounds.