A tetraploid inbred line KAU-TETRA-CL-1 was developed in the watermelon cultivar Sugar Baby through treatment of seedling shoot tips with colchicine solution (0.5%) followed by selection for increased fertility in the C2, C3, C4, and C5 generations. Chloroplast count and guard cell measurement in successive generations of tetraploids were employed as criteria for detecting higher ploidy level and tetraploidy was confirmed by flow cytometry in the C5 generation. A distinct difference was observed in the stomata of diploid Sugar Baby (length: 20.52 ± 1.4 µm; diameter: 15.86 ± 1.0 µm; number of chloroplasts: 11.2 13.1) and tetraploids from the C5 generation (length: 29.12 ± 2.0 µm; diameter: 22.0 ± 0.1 µm; number of chloroplast: 20 22). These parameters were effective in identification of putative tetraploids, and selection based on number of seeds/fruit (>100) was found effective in improving seed number in advanced generations. The tetraploid line selected in the C5 generation (KAU-CL-TETRA-1) produced red-fleshed and yellow-fleshed seedless fruits when used as a female parent with diploid male parents CL-4 and CL-5. A seed yield of more than 200 per fruit was observed in crosses involving the diploid lines. The mean yield over two seasons was 12.256 kg fruits/plant for the red-fleshed KAU-CL-TETRA-1 × CL-4 and 10.168 kg fruits/plant for the yellow-fleshed KAU-CL-TETRA-1 × CL-5 seedless hybrids. The triploid hybrids have excellent fruit quality compared to diploids in terms of sweetness and absence of seeds.