The tropical perennial clover, T. semipilosum, and the species, T. repens, exhibited both similarities and differences in their nodulation characteristics following Rhizobium inoculation. Both T. semipilosum and T. repens were infected via root hairs, formed indeterminate nodules and commenced active nitrogen-fixation approximately 1 week after inoculation. Effective Rhizobium strains produced on average four nitrogen-fixing nodules per T. semipilosum plant, predominantly in the upper region of the tap root. In contrast, 65% of the average 15 nodules per T. repens plant were induced on lateral roots. The Rhizobium strain-ANU843, effective (Nod+Fix+) on T. repens, caused root hair branching and twisting on T. semipilosum, although plants did not fix nitrogen, indicative of an ineffective host–strain interaction. Such ineffective interactions were associated with increased total numbers of nodule-like structures on T. semipilosum; these nodules were atypically associated with, or in close proximity to, lateral roots. Using a GUS gene-modified rhizobia to monitor nodule occupancy, a third of the total nodule-like structures formed from the incompatible interactions showed a positive GUS reaction, indicating that colonization by Rhizobium strains of some of the nodule-like structures had occurred. Moreover, about 70% of the GUS positive nodule-like structures from the incompatible interactions were localized at the junction between the tap and lateral roots. These observations are consistent with an atypical infection route by the incompatible strains.