A greenhouse experiment was conducted to evaluate the genetic relatedness between three Slovenian sweetpotato cultivars; and to assess the effects of different growing substrates on selected agronomic and nutritional traits. Tubers of three cultivars ('Lučka', 'Janja' and 'Martina') with different skin/flesh color were produced in planters under glasshouse conditions in five different growing substrates (perlite, peat, expanded clay, vermiculite and garden soil) from prior raised seedlings. Genetic analysis was performed using a set of eight SSR markers. According to Nei's genetic distance and pairwise population Fst analysis, the most related cultivars are 'Janja' and 'Martina'. The following agronomic traits were evaluated: vine length, thickness of vine-base, number of branches, weight of above ground part, number of leaves plant −1 , number of tubers plant −1 and tubers weight plant −1. Among nutritional traits, total phenolic content (TPC), antioxidant potential (AOP) and ascorbic acid content (AA) were determined. Significant interactions of growing substrates (factor A) × cultivar (factor B) were observed for thickness of vine-base, weight of above ground part, AOP, TPC and AA. Overall results show different response of cultivars in different growing substrate. Growing substrate provide a discriminant classification of the sweetpotato cultivars according to their agronomic and nutritional traits.