The aim of this study was to evaluate the composition of aqueous extracts of in vitro shoot cultures and wild plants of Pterospartum tridentatum in order to promote the use of this plant material as a possible source of bioactive compounds. The extraction yield from wild plants compared with in vitro shoot cultures was lower. The total phenolic contents of in vitro shoots were significantly lower compared with those of wild plants. The phenolic profiles of in vitro shoots were very similar to those of wild plants, regardless of the source. However, taxifolin-6- C-glucoside, as well as rutin and isoquercitrin, were not present in extracts of in vitro shoots. An interesting result was the higher molar percentages of rhamnose and uronic acids detected in in vitro shoots compared with the wild plants, which can make the in vitro plant material very useful for obtaining these compounds.