Sugarcane (Saccharum spp. hybrids) is a major row crop in Florida. The effect of row-spacing on total row length per hectare can significantly effect the cost of major farm operations, including planting, harvesting and fertilizer application. In Florida, sugarcane is commonly planted at 1.5 m row-spacing. There is little information available on the effect of relatively narrow or wide spacing on the growth and yield of sugarcane, especially in new high-biomass cultivars. To determine sugarcane growth response to different row-spacing, a field trial was conducted in organic soil (Histosols) with four different row-spacings (1.2, 1.5, 1.8 m and a double row on a 1.5 m bed) and three sugarcane cultivars (CP 96-1252, CP 00-1101 and CP 01-1372). Data showed differential row-spacing effects on plant cane versus the first ratoon. In plant cane, depending on the cultivar, double row and 1.2 m spacing produced 17–46% and 23–42% greater sugar yield per hectare (TSH) than the current 1.5 m spacing, respectively. Overall, the combined average TSH of plant cane and the first ratoon in double and 1.2 m spacing was 20% and 26% greater than 1.5 m spacing. However, the yields in 1.5 m and 1.8 m row-spacing were similar. There was no significant effect in the first ratoon. There was no row-spacing effect on sucrose concentration. A similar response of cultivars to the tested row-spacings suggests that selecting cultivars for different row-spacings may not potentially show any yield improvements. Compared to the current 1.5 m spacing, narrow-spacing (1.2 m and double row) may potentially increase yields and wide (1.8 m) spacing reduce the production cost but further research is needed to understand the economic feasibility of different row configurations.