High sugar content is a valued papaya fruit quality trait that is difficult to select due to a lack of knowledge regarding the major contributing genetic components and the potential for large environmental effects. The initial step towards better understanding this complex trait is to identify the functional genes involved in the sugar synthesis and accumulation pathways. This was achieved in the current study through differential expression analyses of a suit of sweetness-related genes among two phenotypically contrasting papaya cultivars. The major sugar detected in leaf and ripening fruit tissues of two genotypes (120 days after anthesis to colour break 100%) was sucrose, which accounted for 40-60% of the total sugars detected. In general, genotype 'Sunrise Solo' had higher sugar content than genotype 'RB2' in all tissue types assessed. Subsequently, differential expression of 11 genes potentially associated with the main sugar synthesis and accumulation pathways were investigated. Of these, sucrose phosphate synthase (cpSPS1, cpSPS2, cpSPS3 and cpSPS4) and invertase (cpCWINV1and cpAVIN2) gene family members were expressed significantly differently. In all tissue types, cpSPS2 was between 0.5 to 3 times more highly expressed in 'Sunrise Solo' than 'RB2'. The maximum expression of cpSPS2 in 'Sunrise Solo' was observed in mature fruit at 120 days after anthesis, while its expression remained constant in 'RB2'. Similarly, genes cpCWINV1 and cpAVIN2 were significantly more expressed in 'Sunrise Solo' compared to 'RB2' during ripening stages. Further validation and investigation of the expression of these gene sequences for association with the sweetness trait at specific fruit ripening stages and in different environments will aid in their identification as candidate sweetness markers for future selective breeding strategies.