Introduction The Cactaceae family exhibits remarkable adaptation to arid environments, yet mitochondrial genomic resources remain extremely scarce for this lineage. Lophophora williamsii (peyote) is an ecologically and culturally significant cactus species native to the Chihuahuan Desert, but its mitochondrial genome has never been characterized.Methods We assembled and annotated the first complete mitochondrial genome of L. williamsii using PacBio HiFi long-read sequencing, and conducted comprehensive analyses including repeat identification, codon usage, RNA editing prediction, intracellular gene transfer detection, phylogenetic reconstruction, selection pressure assessment, and synteny comparison.Results The assembled mitogenome is exceptionally large (2,422,778 bp) and structurally complex, comprising 57 contigs that resolve into five linear molecules. It encodes 33 core and 13 variable protein-coding genes, three rRNA genes, and 49 tRNA genes. A total of 587 simple sequence repeats (SSRs), 124 tandem repeats, and 5,519 interspersed repeats (>= 30 bp) were identified. Codon usage bias is predominantly driven by natural selection. We predicted 431 C-to-U RNA editing sites across 32 PCGs and detected 18 chloroplast-derived DNA fragments (31,614 bp) containing 19 intact genes. Phylogenetic analysis based on 30 conserved mitochondrial PCGs placed L. williamsii as sister to Mammillaria huitzilopochtli. Most mitochondrial genes are under purifying selection, whereas atp8 and matR exhibit positive selection. Synteny analysis revealed extensive collinear blocks decreasing with phylogenetic distance.Discussion This first complete mitogenome of L. williamsii provides a fundamental genomic resource for understanding mitochondrial evolution in Cactaceae, offers high-resolution molecular markers for species identification and forensic authentication, and supports conservation genetics for this threatened species.