Four different low molecular weight (LMW) RNA profiles, designated I-IV, among 179 isolates from Medicago, Melilotus and Trigonella species growing in a field site in Northern Spain were identified. From sequence analysis of the 16S rRNA, atpD and recA genes as well as DNA-DNA hybridization analysis with representatives of each LMW RNA profile it was evident that isolates with LMW RNA profiles I and II belonged to Sinorhizobium meliloti and those displaying profiles III and IV to Sinorhizobium medicae. Therefore, two distinct LMW RNA electrophoretic mobility profiles were found within each of these two species. Collectively, LMW RNA profiles I and II (identified as S. meliloti) were predominant in Melilotus alba, Melilotus officinalis and Medicago sativa. Profiles III and IV (identified as S. medicae) were predominant in Melilotus parviflora, Medicago sphaerocarpa, Medicago lupulina and Trigonella foenum-graecum. All the four LMW RNA profiles were identified among isolates from Trigonella monspelliaca nodules. These results revealed a different specificity by the hosts of the alfalfa cross-inoculation group towards the two bacterial species found in this study.