Context. The Gran Chaco is a livestock producing region of Argentina with several under-utilised legume forage genetic resources, including species from the genus Macroptilium.

Aims. We aimed to generate reliable characterisation and selection schemes in germplasm of Macroptilium for biological nitrogen fixation with native rhizobia, and evaluate the germplasm variability for initial growth and nodulation capacity with native soil of subtropical environment.

Methods. The trial evaluated 20 accessions of four Macroptilium species with or without added nitrogen. The variables were analysed through a nested ANOVA with the factor species, accessions nested within species, nitrogen level, and the corresponding interactions. Relationships between variables and accessions were examined using principal component analysis.

Key results. There was variability in the symbiotic response and initial growth, and most of the variance was explained by differences between species. Nodulation capacity was related with biological nitrogen fixation, since the accessions with higher nodule number and nodule weight showed higher initial growth and higher crude protein content in shoot.

Conclusions. We found variability for nodulation capacity associated with biological nitrogen fixation within the evaluated germplasm of Macroptilium. In relation to effectiveness, five of 20 accessions evaluated were classified as effective.

Implications. Our breeding approach could contribute to develop native legume forage cultivars with better symbiosis with native rhizobia, which would reduce implantation costs and reduce the use of chemical nitrogen fertilisers.