Purpose <p>Superfecundation, the fertilization of two oocytes by different spermatozoa within the same ovulatory cycle, can result in monopaternal or heteropaternal dizygotic twins. While monopaternal superfecundation is more common, heteropaternal superfecundation is rare and typically seen in disputed paternity cases. This study presents a case of heteropaternal superfecundation confirmed through forensic DNA analysis and reviews its occurrence in existing literature.</p> Methods <p>A forensic investigation was conducted in a court-ordered paternity case involving dizygotic twins, their mother, and an alleged father. Buccal swab samples were collected and analyzed using multiplex amplification of 19 STR markers and the amelogenin locus. A second DNA test confirmed the results. Additionally, a dataset of 2,679 paternity tests over 10 years was examined to estimate paternity exclusion rates in twin cases.</p> Results <p>Genetic analysis confirmed the alleged father’s paternity of twin 1 but not twin 2, with 14 out of 19 STR loci showing absent alleles in twin 2. The 10-year dataset showed 553 paternity exclusions (20.64% of cases), with 31 involving twins, of which one case (3.23%) was identified as heteropaternal superfecundation. No significant difference was found between paternity exclusion rates in twin and non-twin cases.</p> Conclusions <p>This case underscores the value of forensic genetic testing in detecting heteropaternal superfecundation, a rare occurrence with legal and social implications. Advances in DNA analysis may lead to more frequent identification of such cases.</p>