Polyacetylenes (PAs) are a large group of bioactive phytochemicals, which are primarily produced by higher plants of the families Apiaceae and Araliaceae. Especially aliphatic C17-polyacetylenes of the falcarinol-type such as falcarinol (FaOH) and falcarindiol (FaDOH) are known for their numerous biological functions including positive effects on human health. In this study we investigate the potential of carrot hairy root (HR) cultures for accumulation of PAs in vitro. Three individual plants of seven differently colored carrot cultivars were used for the development of HR cultures by transformation of root discs with the wild-type Rhizobium rhizogenes strain 15834. A total of 51 individual HR lines were established and quantitatively analysed by HPLC/DAD together with root, petiole and leaf tissue samples for their FaOH and FaDOH levels. Among the five tissues sampled from the donor plants, root periderm samples generally exhibited the highest PA levels with FaDOH as prevailing PA and large differences between cultivars. Compared with the periderm, FaOH levels were highly increased in HRs of all investigated carrot cultivars, whereas the FaDOH levels were only slightly enhanced. Considering the low to moderate PA concentration in root and leaf tissues of the orange cultivars there was an up to 30-fold increase of the FaOH concentration in HRs derived from orange genotypes. In this study, we demonstrate, that carrot HRs are able to produce large amounts of PAs. Hence, we suggest that HRs might be a suited tool to assess the function of candidate genes involved in the biosynthesis of major PAs.