A heteropaternal male twin case with two men being alleged fathers was investigated as requested by the Court. Up to 37 PCR-based polymorphic DNA systems were studied in this case which was complicated by a paternal ACTBP2 mutation detected in one twin. This is the first report on a STR mutation in a double paternity case where both biological fathers were indisputably identified. The STR systems enable the resolution of these complex genetic relationships even in a case where a mutation in one STR locus was encountered.