A total of 43 Rhizobium leguminosarium bv. trifolii isolates were obtained from soil samples of two ball clover (Trifolium nigrescens) pastures from Iola and Kilgore (Texas) using ball clover as capture plants. The isolates were phenotypically characterized by intrinsic antibiotic resistance (IAR) against a range of concentrations of eight antibiotics, and by the utilization of 95 different carbon sources (BIOLOG system). The rhizobial isolates were also evaluated for their tolerance to salinity, high temperatures and low pH. The isolates showed two different ranges of growth rates: fast-growing (doubling times between 1.4 - 3.7 h) and slow- growing isolates (12.3 - 21.3 h). The numerical analysis of the phenotypic characteristics showed that the 43 isolates could be grouped in 24 different strains. Cluster analysis based on sensitivity responses of IAR, metabolic profiles of BIOLOG and salt, temperature and acidity tolerance levels could distinguish the Rhizobium strains from a Pseudomonas isolate. The analysis also showed that the rhizobial strains isolated from ball clover nodules are different from a commercial R. leguminosarium bv. trifolii strain used as inoculant for this legume.