Steroidal glycoalkaloids occur in potatoes and are reported to impart resistance to phytopathogens including bacteria, fungi, and insects. Because glycoalkaloids can be passed to progenies during breeding programs designed to develop improved potatoes, it is of importance to determine the quality of desired characteristics and the composition of glycoalkaloids of new somatic hybrids. The objective of this study was to determine the appearance, size, and shape (morphological characters) as well as the glycoalkaloid content of potato tubers of somatic hybrids between tetraploid Solanum tuberosum cv. Dejima (2n = 4x = 48 chromosomes) and the dihaploid clone ATDH-1 (2n = 2x = 24 chromosomes) induced by anther culture from Solanum acuale-T (acl-T, 2n = 4x = 48 chromosomes). Tuber size and shape in somatic hybrids were in accord with those of cv. Dejima, whereas the tuber skin color resembled that of ATDH-1. Thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography/mass spectrometry studies showed that the two steroidal glycoalkaloids (alpha-chaconine and alpha-solanine) were present in the tubers of S. tuberosum, whereas acl-T and ATDH-1 tubers were found to contain alpha-tomatine and demissine. The concentrations of total glycoalkaloids in both acl-T and ATDH-1 was >100 mg/100 g of fresh weight tuber cortex, much higher than in S. tuberosum. All somatic hybrids, except one clone, contained four glycoalkaloids (alpha-chaconine, alpha-solanine, alpha-tomatine, and demissine) derived from the fusion parents. The lack of alpha-tomatine in the remaining clone may be due to somaclonal variation. The results show that character expression is influenced by ploidy level and that total glycoalkaloid levels in most somatic hybrids were intermediate between those of the fusion parents. The possible significance of these findings for plant breeding and food safety is discussed.