Low-molecular-weight RNA analysis was performed for the identification and classification of 20 Argentinian strains isolated from the root nodules of Prosopis alba. SDS-PAGE of total cellular proteins, determination of the DNA base composition, DNA-DNA reassociation experiments and physiological and biochemical tests were also carried out for these strains and the whole 16S rRNA gene was sequenced from one of the strains, strain LMG 19008T. Results of the genotypic and phenotypic characterization showed that the strains isolated in this study belong to a group that clustered in the genus Mesorhizobium. The results of DNA-DNA hybridizations showed that this group is a novel species of this genus. The name Mesorhizobium chacoense sp. nov. is proposed for this species. The type strain is LMG 19008T (= CECT 5336T).