Cactaceae plant family comprises over 130 genera and 2000 species of succulent flowering plants. The genera Mammillaria and Notocactus (Parodia), which have medicinal and nutritional applications as well as aesthetic appeal, are considered to be among the major genera of the family. Several species of both genera show morphological and chemical similarities and diversities according to environmental conditions and genotypes. Here, we assessed the genetic relationships of nine species belonging to two major genera Mammillaria and Notocactus under the family Cactaceae, using two modern gene-targeting marker techniques, the Start Codon Targeted (SCoT) Polymorphism and the Conserved DNA-Derived Polymorphism (CDDP). Besides, we screened the various phytochemicals and evaluated the antioxidant activities of the nine species of cacti. Five out of the 10 SCoT and eight CDDP primers used to screen genetic variations within the nine species yielded species-specific reproducible bands. The entire 156 loci were detected, of which 107 were polymorphic, 26 were monomorphic, and 23 were unique loci. The nine species were categorized into two groups based on the dendrogram and similarity matrix. Phytochemical profiling revealed that sterols, triterpenes, flavonoids, and tannins were found in all the tested species. Additionally, two Notocactus species (N. shlosserii and N. roseoluteus) and one Mammillaria species (M. spinosissima) revealed a considerable antioxidant activity. Our results demonstrated that gene-targeting marker techniques were highly powerful tools for the classification and characterization of the nine investigated species, despite displaying high similarities at both morphological and phytochemical levels.