Mutations are the key drivers for evolution and diversification in plants. In varietal selection, sources for variation are always sought as starting breeding materials. Thus, in the absence of desired natural variations in breeding populations, targeted or random mutagenesis is applied to induce variations. Cultivated potato (Solanum tuberosum L.) is autotetraploid crop species with a narrow and highly heterozygous genetic base, and the complexity of its genome makes its genetic studies more difficult. In the current study, induced mutagenesis was performed in diploid potato using ethyl methane sulfonate (EMS) to enlarge the genetic variability for its use as pre-breeding materials in both polyploid and diploid potato breeding. As starting materials, true potato seeds were treated with 1.2% EMS for 4–6 h along with untreated seeds as controls. A large variation in terms of germination rate, plant, flower, and tuber phenotype was observed in EMS-treated plants compared with their untreated counterparts. In particular, abnormal phenotypes including twisted stem, partial and (or) completely chlorotic leaves and stems, variations in stem colour and weak-stemmed plants with lateral growth habit as well as plants with determinate growth habit were observed along with normal plant characteristics. Moreover, variations in flower colour and tuber colour, shape, and size, as well as yield potential, were observed in EMS-treated lines. The reported phenotypic characterization of EMS mutagenized diploid potato collection is to our knowledge the first in its kind and represents a premium genetic resource for potato breeding programs and plant biologists for genes functional characterization in potato.