Bacterial strains isolated from nitrogen-fixing nodules of Lupinus mariae-josephae have been characterized following genetic, phenotypic and symbiotic approaches. Analysis of 16S rRNA genes placed them in a group together with Bradyrhizobium elkanii USDA 76 T , B. pachyrhizi PAC48 T , B. jicamae PAC68 T , ‘B. retamae’ Ro19 T and B. lablabi CCBAU 23086 T with over 99.0% identity. Phylogenetic analysis of concatenated housekeeping genes, recA, atpD and glnII, suggested that L. mariae-josephae strains represent a new Bradyrhizobium species, closely related to B. lablabi CCBAU 23086 T , B. jicamae PAC68 T and ‘B. retamae’ Ro19 T with 92.1, 91.9 and 90.8% identity, respectively. These results are consistent with overall genomic identities calculated as Average Nucleotide Identity (ANIm) using draft genomic sequences obtained for relevant strains. While L. mariae-josephae strains LmjM3 T /LmjM6 exhibited a 99.2% ANIm value, they were significantly distant (<93% ANIm) from type strains of their closest species (‘B. retamae’ Ro19 T , B. lablabi CCBAU 23086 T and B. jicamae PAC68 T ). Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (WC-MALDI-TOF-MS) analysis of proteomic patterns of the same strains was consistent with these results. The symbiosis-related genes nodC, nodA and nifH genes from strains nodulating L. mariae-josephae were phylogenetically related to those from ‘B. retamae’ Ro19 T , but divergent from those of strains that nodulate other lupine species. Based on genetic, genomic, proteomic and phenotypic data presented in this study, L. mariae-josephae nodulating strains LmjM3 T , LmjM6 and LmjM2 should be grouped within a new species for which the name Bradyrhizobium valentinum sp. nov. is proposed (type strain LmjM3 T =CECT 8364 T , LMG 2761 T )