We report here the isolation and characterisation of two new β-1,3-glucanase cDNAs, SrGLU3 and SrGLU4, from a tropical legume Sesbania rostrata Bremek. & Oberm., which form N2-fixing nodules on the stem after infection by Azorhizobium caulinodans. SrGLU3 was characterised as being grouped in a branch with tobacco class I β-1,3-glucanases, where the isoforms were reported to be induced by either pathogen infection or ethylene treatment. SrGLU4 was characterised as separate from other classes, and we propose this new branch as a new class (Class VI). The SrGLU3 gene was constitutively expressed in normal stem nodules induced by the wild type strain of A. caulinodans (ORS571), and also even in immature stem nodules induced by a mutant (ORS571-C1), which could not form mature stem-nodules. In contrast, the transcript accumulation of SrGLU4 was hardly detectable in immature nodules inoculated by the ORS571-C1 mutant. We suggest that S. rostrata makes use of SrGLU4 to discriminate between symbionts and non-symbionts (mutants) in developing nodules. We propose the SrGLU4 gene as a new nodulin during nodulation.