Abstract The aim of the present study was to characterize rhizobia isolated from the root nodules of Trigonella foenum-graecum (fenugreek) cultivated in the sub-himalayan region of Uttarakhand by means of morphological, physiological, biochemical and molecular characteristics to know their phylogenetic relationships. The genetic relatedness amongst twenty root nodule isolates of fenugreek was analyzed employing PCR-RFLP (ARDRA) analysis of 16S rDNA amplicons, ERIC-PCR and sequencing technique. Restriction analysis of the amplified 16S rRNA gene and ERIC-PCR of fenugreek rhizobia led to the identification of three ribotypes that appear to represent three distinct species: Ensifer (Sinorhizobium) meliloti, Rhizobium leguminosarum and Burkholderia sp. Based on plant growth promoting activities four isolates RHT2, RHT8, RHT12 and RHT19 were selected from three distinct groups and identified as Ensifer (Sinorhizobium) meliloti , Burkholderia sp., Burkholderia sp., Rhizobium leguminosarum by 16S rDNA sequences. The majority of isolates is closely related to Ensifer (Sinorhizobium) meliloti. This study clearly shows that the characterization of symbionts of fenugreek will reveal additional diversity. Highlights • Genetic diversity among microorganisms has been widely studied by PCR-based techniques, which involve the use of primers based on conserved regions of the rRNA genes and digested with a combination of resriction enzymes and/or on short repetitive elements present in the bacterial genome, ERIC (enterobacterial repetitive intergenic consensus), which yields genomic fingerprints specific to pathovars and strains of Gram-negative bacteria. The ERIC profiles are specific for bacterial strains within a species. 16S rDNA genes are ubiquitous in living organism and, therefore, 16S rDNA operon are present in multiple copies and amplimers generated by PCR ribotyping indicate inter and intra-species genetic diversity in the number and structure of ribosomal operon. Following methods were commonly used to study the genetic diversity among bacterial isolates. Amplified Ribosomal DNA Restriction Analysis (ARDRA) and Enterobacterial repetitive consensus sequences (ERIC). • Restriction analysis of the amplified 16S rRNA gene and ERIC-PCR of fenugreek rhizobia led to the identification of three ribotypes that appear to represent three distinct species: Ensifer meliloti, Rhizobium leguminosarum and Burkholderia sp. and same was confirmed by 16S rDNA sequences. The majority of isolates is closely related to Ensifer meliloti. • High diversity of nodulating rhizobia was found which provides sufficient effective strains of indigenous rhizobia that may play both direct and indirect roles in promoting plant growth and disease suppression in Trigonella foenum-graecum. [ABSTRACT FROM AUTHOR]