Spontaneous polyploids are expected in the genus Kaempferia, and polyploidization could be a pivotal factor to promote speciation in this genus. K. galanga Linn. var. latifolia Dunn, a variety of K. galanga Linn., is morphologically different from K. galanga var. galanga, and we hypothesize that various ploidy levels are responsible for their differences. Furthermore, this work aims at exploring the phenotypic traits associated with different level of ploidy. Flow cytometry (FCM) was used to measure cytotypes, and the results indicate that K. galanga var. latifolia is tetraploid while K. galanga var. galanga is pentaploid. One-way analysis of variance (ANOVA) was used to characterize the differences in phytochemical contents as well as 27 morphological traits from leaves, stomatas, and flowers. Comparative analyses of morphological characters showed that polyploidization significantly affected some traits; tetraploid individuals had wider leaves, and pentaploid individuals displayed a higher stomatal density, wider lateral staminodes, longer bracteoles, and bracts (p < 0.05). Another interesting result showed that pentaploid individuals had higher (E)-Ethyl-p-methoxycinnamate content in their rhizomes but lower content in their leaves compared to tetraploid plants (p < 0.05). In conclusion, polyploidization plays a significant role in phenotype differences between tetraploid K. galanga var. latifolia and pentaploid K. galanga var. galanga. Moreover, data on morphology and phytochemicals will offer some references for plant identification and breeding in K. galanga.