Heteropaternal superfecundation is an extremely rare phenomenon that occurs when a second ova released during the same menstrual cycle is additionally fertilized by the sperm cells of a different man in separate sexual intercourse.In August, 2018, the Grupo de Genética de Poblaciones e Identificación at Universidad Nacional de Colombia received a request to establish the paternity of a pair of male twins with genetic markers. The following analyses were performed: amelogenin gene, autosomal short tandem repeat (STR), and Y-STR analyses by means of human identification commercial kits, paternity index, and the probability of paternity calculation and interpretation. A paternity index of 2.5134E+7 and a probability of paternity of 99.9999% for twin 2 were obtained while 14 out of 17 Y-chromosome markers and 14 out of 21 autosomal short tandem repeats were excluded for twin 1. The results indicated that the twins have different biological fathers.Although heteropaternal superfecundation is rarely observed among humans given its low frequency, in paternity disputes for dizygotic twins it is mandatory to demand the presence of the two twins in the testing to avoid wrong conclusions.