In order to investigate the effects of homogeneous and localized supply of different nitrogen forms (nitrate, NO3− vs ammonium, NH4+) on the growth of tomato seedlings, root morphology and six cytokinin (CTK) fractions in xylem sap were analyzed. Whole roots were supplied with different ratios of NO3− to NH4+ (100:0, as 100-0NA; 75:25, as 75-25NA; 50:50, as 50-50NA) under homogeneous supply. In split-root experiments, three treatments were compared: a sole NO3− supply (N|N), a spatially separated supply of NO3− and NH4+ (N|A), and a spatially separated supply of NO3− and a mixture of NO3− and NH4+ nutrition at a ratio of 75:25 (N|AN). All concentrations of total N were set at 5 mM. The results showed that (1) homogeneous 75% NO3− plus 25% NH4+ supply to the whole root zone led to maximum shoot and root dry matter (DM), root surface area (RS) and root volume (RV). The spatially separated supply of NO3− and NH4+ (N|A) resulted in a contrasting effect on root morphology: in comparison to N|N, root DM in the NO3−-containing pot was increased by 50% whereas it was depressed by 50% in the NH4+-containing pot. The 75% NO3− plus 25% NH4+ supply in the split-root experiment led to no significant effects either on shoot DM and root DM, or on RS and RV when compared to N|N. (2) The presence of NH4+ in the external medium led to a significantly reduced total xylem-CTK concentration, and a close negative correlation was found between xylem NH4+ and total CTK concentration irrespective of culture mode. A relatively high level of zeatin riboside (ZR) was maintained both in 75-25NA and N|A treatments. It was concluded that, in addition to the percentage of NH4+ to NO3− in the nutrient solution, whether NH4+ was supplied to the whole root system or to only part of the root system was also an important factor affecting plant growth. The fact that the 75-25NA and N|A treatments resulted in optimal growth of tomato seedlings might be attributed to the higher ZR concentration in xylem.