The oxalate content was assessed in nine diverse genotypes of cherry tomato in ripened fruit, leaf and root tissues. The maximum oxalate accumulation was found in leaf lamina followed by mature fruit and root tissues among all genotypes where the lowest oxalate content was found in the genotype FB-3-5 and the highest content was recorded in the genotype RSC-4. Examining the leaf and root of nine cherry tomato genotypes at three developmental stages, the investigation into the biosynthesis of oxalates focused on the study of two enzymes, glycolate oxidase (GLO) and ascorbate peroxidase (APX). A positive correlation was observed between oxalate formation and enzyme specific activity of both enzymes. Examining the relative gene expression of oxalate biosynthesizing genes, SlGLO2 and SlAPX7, indicated that the gene SlGLO2 encoded the GLO enzyme and played a crucial role in oxalate biosynthesis in cherry tomatoes. Understanding oxalate biosynthetic pathways and gene expression will aid in developing mechanisms to enhance cherry tomato nutritional quality and improving breeding programmes.