Supplementary lighting is commonly used in high-quality seedling production. In this study, grafted tomato seedlings were grown for 10 days in a glasshouse with a 16-hour daily supplementary lighting at 100 μmol·m⁻²·s⁻¹ PPFD from either high pressure sodium (HPS), metal halide (MH), far-red (FR), white LEDs (W), or mixed LEDs (W₁R₂B₁) to determine which light sources improve the seedling quality. The control did not have any supplementary light. The physiological parameters and the expression of genes related to photosynthesis were analyzed. The results show that the root length, biomass, number of leaves, chlorophyll (SPAD), dry weight to height ratio (WHR) of the scion, and specific leaf weight (SLW) were the greatest for grafted seedlings grown in W₁R₂B₁. The level of root ball formation was the greatest for seedlings grown in W₁R₂B₁, followed by those grown in W, HPS, and MH. Seedlings grown in FR did not fare very well, as they were very thin and weak. Moreover, the expression of two photosynthetic genes (PsaA and PsbA) was significantly increased by W₁R₂B₁ and W, which suggests that the plastid or nuclear genes might be regulated. The overall results suggest that W₁R₂B₁ was the most suitable light source to enhance the quality of grafted tomato seedlings. The results of this study could be used as a reference for seedling production in glasshouses, and provide new insights in the research of lights affecting the development of plants.