[Background] Research on rhizobium diversity has paved the way for utilization of rhizobial germplasm resources. [Objective] To research the phenotypic and genetic diversity of endophytic and non-endophytic rhizobia of alfalfa (Medicago sativa L.), and verify the hypothesis that rhizobial symbiotic efficiency differed according to alfalfa variety by comparing their symbiotic difference on five alfalfa varieties. [Methods] Endophytic (seed, flower, leaf, stem, root epidermis, root stele, nodule) and non-endophytic (rhizosphere soil and field soil) bacteria isolates were collected from M. sativa cvs. Longzhong and Qingshui in arid crop area of Huining, Baiyin, M. sativa cv. WL168HQ in irrigated area of Anning, Lanzhou, and M. sativa cvs. Gannong No. 3 and Gannong No. 9 in irrigated area of Liangzhou, Wuwei, Gansu. Numerical analysis, 16S rRNA restriction fragment length polymorphism fingerprinting (RFLP), 16S rRNA gene sequencing, multilocus sequence typing (MLST) of concatenated sequences of atpD, glnII, and recA genes, and sequence analysis of symbiotic genes nodC and nifH were applied to study the phenotypic and genetic diversity of endophytic and non-endophytic rhizobia. A principal component analysis (PCA) was used to investigate their symbiotic differences on five alfalfa varieties as well. [Results] Totally 43 endophytes and 10 non-endophytic isolates were obtained. None were collected from flowers and leaves. The phenotypic diversity of these 53 isolates along with two reference strains (R.GN5 and S.12531) were abundant, with eight phenotypic clusters formed. Twenty-two RFLP patterns were produced after 16S rRNA-RFLP analysis, and the most widespread genotype among the isolates was that designated as genotype Ⅰ (24). Three other genotypes (Ⅻ, ⅩⅤ and ⅩⅨ) occurred less frequently in alfalfa symbionts (five, five and three). There were 16 genotypes specific to a single M. sativa isolate. According to the phylogenetic analyses of 16S rRNA gene and MLST, isolates were further classified into Rhizobium radiobacter, R. rosettiformans, and Ensifer meliloti. The nodC and nifH gene fragments were only amplified and sequenced from seven representative E. meliloti strains and reference strain S.12531, indicating that they were capable of nodulating alfalfa. The nodule number per plant, shoot dry weight and crude protein content of M. sativa cvs. Gannong No. 3 (inoculated with G3L3), Longzhong (inoculated with LP3, LL1 and LL2), Qingshui (inoculated with QL2), and WL168HQ (inoculated with LL1, LL2 and WLP2) were promoted simultaneously. The parameter values of M. sativa cvs. Gannong No. 3, Gannong No. 9, and Qingshui plants inoculated with the E. meliloti isolates clustered together, which ranged from ?1 to 1 in PC1 axis and ?1.5 to 1.5 in PC3 axis. Compared with these three alfalfa varieties, that of M. sativa cvs. Longzhong and WL168HQ plants dispersed greatly and ranged from ?1.5 to 4 in PC1 axis and ?3 to 4 in PC3 axis. [Conclusion] The phenotypic and genetic diversity of endophytic and non-endophytic rhizobia were abundant, and there was no direct relationship between diversity and strains’ origins. Strong mutualistic symbiosis and adaptability were presented between G3L3 and M. sativa cv. Gannong No. 3, LP3, LL1, LL2 and M. sativa cv. Longzhong, QL2 and M. sativa cv. Qingshui, and LL1, LL2, WLP2 and M. sativa cv. WL168HQ. The tested strains exhibited similar symbiotic efficiency when inoculated onto M. sativa cvs. Gannong No. 3, Gannong No. 9, and Qingshui plants, while an obvious symbiotic difference of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants. Their symbiotic efficiency varied according to alfalfa varieties, which manifested that the sensitivity of different alfalfa varieties to rhizobial strains may differ.