Current evidence supports a single pathway of oxalate biosynthesis utilising ascorbic acid as the precursor. In this study, we begin to address the possibility that more than one pathway of oxalate biosynthesis and calcium oxalate formation occurs in Medicago truncatula Gaertn. (cv. Jemalong genotype A17). Like the wild type, developing leaves of the calcium oxalate defective (cod) 4 mutant contain prismatic crystals along the vascular strand, but this mutant also hyper-accumulates druse crystals within the mesophyll cells. A second mutant, cod5, fails to accumulate prismatic crystals along the vascular strand, but is capable of wild type druse crystal accumulation in maturing leaves. To assess whether a single pathway of oxalate biosynthesis and calcium oxalate formation occurs in M. truncatula, we generated and characterised the cod4/cod5 double mutant. Microscopic examination of the cod4/cod5 revealed that the double mutant exhibits both cod4 and cod5 mutant crystal phenotypes simultaneously, suggesting there are differences in the pathways leading to the two crystal types. Measured ascorbic acid levels and ascorbate induction studies were consistent with the acid as precursor to oxalate in druse crystal formation but not necessarily prismatic crystal formation. On the basis of these findings, we propose a working model depicting possible pathways of oxalate biosynthesis and calcium oxalate formation.