The distribution of betalains in peel, flesh, and petioles of yellow and red beetroot cultivars has been investigated using an High‐performance liquid chromatography (HPLC) system with electrospray mass spectrometry. Differences in the levels of betacyanins and betaxanthins between different colored cultivars were individually determined for 3 plant parts. The content of almost all analyzed compounds decreased in the following order: peel > flesh > petiole. Betanin/isobetanin pigments comprised a major portion of the relative peak area measured in red beetroot peel. Isobetanin relative peak areas were also high in leaf petioles (68.94% to 74.16%) of red colored cultivars. However, betacyanins were completely absent from the extracts of all analyzed parts of yellow beet. Glutamine‐bx represented a very high relative peak area (59.54% to 64.18%) in flesh of red‐colored cultivars analyzed in the study. Our results indicate that red beet cultivars can be utilized as a potential source of red and yellow natural colorants. However, differences in pigment composition among different beetroot parts must be considered and in order to maximize the pigment yields petioles can also be used as a source rich in specific betalain compounds. Practical Application The studies on plant material with high betalain levels can provide new opportunities for the use of these healthy pigments in the food and pharmaceutical industries. Further investigations on highly concentrated betalains in various cultivars/plants parts can help elucidate their biological activity.