The genus Opuntia belongs to the Cactaceae family. The cladodes, also known as “nopalitos”, have been widely used, in many countries, for their beneficial effects and phytochemical composition. It is used as a food, as a source of nutrients, and also in folk medicine [1]. In the present work, the cladodes of Opuntia macrorhiza (Engelm.) and Opuntia microdasys (Lehm.) were characterized in terms of free sugars, organic acids, fatty acids, tocopherols and phenolic compounds. The individual sugars’ profile was determined by HPLC-RI, organic acids by HPLC-DAD, fatty acids by GC-FID, and tocopherols by HPLCfluorescence. The phenolic compounds profile was determined by HPLC-DAD-MS/ESI. Despite the phylogenetic proximity and similar geographical origin the samples of O. microdasys and O. macrorhiza showed significant differences in the profiles of sugars, organic acids, fatty acids, tocopherols and phenolic compounds. While, O. microdasys presented higher sugars and tocopherols content, O. macrorhiza revealed higher concentration in organic acids, poly and monounsaturated fatty acids. The cladodes of O. microdasys (10 compounds: 6 phenolic acid derivatives and 4 favonols) revealed a higher amount of phenolic compounds in comparison with O. macrorhiza (7 compounds: 6 phenolic acid derivatives and 1 favonol); only four of the identified compounds were detected simultaneously in both species. Piscidic acid (3400±236 μg/g of extract) and eucomic acid (1688±26 μg/g of extract) were the main phenolic compounds in O. macrorhiza, while isorhamnetin-O-(rhamnosyl)-rutinoside was the most abundant in O. microdasys (2507±73 μg/g of extract). Hence, the elucidation of the most abundant compounds might constitute useful information to select the best species regarding specific applications of these natural extracts.