<p>Passion fruit is a tropical fruit with significant economic value. This study represents the first systematic application of start codon targeted (SCoT) markers to assess genetic diversity and population structure in passion fruit germplasm. The SCoT-PCR, including DNA template quantity, primer concentration, and 2 × Taq Plus Master Mix, was optimized using an L₁₆(4³) orthogonal design. From 36 initial primers, 24 core primers with clear and reproducible bands were selected. Using the optimized system, 140 passion fruit accessions were genotyped, yielding 604 bands, of which 599 were polymorphic (98.93%). The average Polymorphism information content (PIC) was 0.247, Nei’s gene diversity index (H) was 0.251, and Shannon’s information index (I) was 0.396, which are lower than the levels of genetic diversity reported in research on passion fruit and its closely related species (such as PIC = 0.657), indicating a moderately low level and suggesting that its genetic diversity is relatively conserved. The unweighted pair group method with arithmetic mean (UPGMA) clustering and principal coordinate analysis (PCoA) yielded consistent results, dividing the 140 samples into 6 major groups. Our results demonstrate that SCoT markers are effective tools for genetic diversity analysis and phylogenetic identification in passion fruit, providing a scientific basis for core germplasm collection, parental selection, and molecular marker-assisted breeding programs, directly supporting the genetic improvement of this crop.</p>