The Crassulaceae family displays extensive morphological diversity and complex evolutionary trajectories across its constituent genera. These attributes significantly challenge the reconstruction of intrafamilial phylogenetic relationships. While chloroplast and nuclear DNA markers are widely used for phylogenetic analyses, mitochondrial DNA (mtDNA) remains underutilized, despite its demonstrable potential as a taxonomic marker. Sequence variation in the mitochondrial nad7 intron was analyzed across six species representing two genera (Phedimus and Sedum) native to Korea. The objective was to assess genetic diversity and determine whether the nad7 intron could effectively augment existing chloroplast and nuclear DNA markers as a taxonomic tool. Target amplicon length spanned 974 to 998 base pairs (bp). Multiple sequence alignment of the six Crassulaceae species from Phedimus and Sedum identified single nucleotide polymorphisms (SNPs), insertions/deletions (INDELs), and dinucleotide polymorphisms, interspersed with conserved nad7 intron regions. Interestingly, an INDEL at positions 189–192 exhibited a diagnostic genus-level pattern, facilitating the unambiguous separation of Phedimus and Sedum. This outcome is consistent with established morphological and chloroplast DNA–based classifications. Collectively, these findings validate the mitochondrial nad7 intron as a promising supplementary molecular marker for taxonomic classification and genetic resource conservation within Crassulaceae.